RU2325202C2 - Conjugates of ghrelin carrier - Google Patents

Abstract

FIELD: molecular biology; virology; immunology; medicine.

SUBSTANCE: invention is related to a composition containing virus-like particle and at least one bound ghrelin or peptide obtained from ghrelin. The invention is also related to the production method of conjugates and well-ordered and recurring massifs. Invented compositions are suitable for vaccine production to treat obesity or other diseases associated with increased consumption of food or body weight. They are also suitable for effective induction of immune responses and antibodies responses, in particular.

EFFECT: invented compositions are suitable for effective induction of auto specific immune responses.

30 cl, 3 dwg, 21 ex

Description

BACKGROUND OF THE INVENTION

Field of Invention

The present invention relates to the fields of molecular biology, virology, immunology and medicine. The invention relates to a composition containing an array of ordered and repeating antigens or antigenic determinants, and in particular an array of ghrelin or peptide derived from ghrelin. More specifically, the invention relates to a composition comprising a virus-like particle and at least one ghrelin or peptide derived from ghrelin associated with it.

The invention also relates to a method for producing conjugates and ordered and repeating arrays, respectively. The compositions of the invention are suitable for the preparation of vaccines for the treatment of obesity and other diseases associated with increased food intake or increased body weight, and for the effective induction of immune responses, in particular antibody responses. In addition, the compositions of the invention are particularly suitable for efficiently inducing an autospecific immune response within the context of this context.

Linked Area

Obesity is a disease that affects millions of people around the world. Although the underlying causes of obesity may be different, a common cause for almost all forms of obesity is increased food intake. Hunger and eating behavior are regulated by many factors, including leptin, growth hormone, neuropeptide Y (NPY), related agouti protein (AGRP) and others. A recently identified key regulator of eating behavior is ghrelin, an acylated peptide produced in the stomach as well as in certain parts of the brain (hypothalamus) (Kojima et al. Nature 402: 656-660 (1999)). Ghrelin is formed by enzymatic cleavage from a precursor form of a gap consisting of 117 amino acids with the formation of a peptide of 28 amino acids with n-octanoylation at serine 3. n-Octanoylation at this position is necessary for the biological activity of ghrelin. The second isoform of ghrelin from 27 a.k. (ghrelin-desQ14) with glutamine (Q) absent at position 14, however, this isoform is only an insignificant component of circulating ghrelin. Like the full-sized ghrelin, the biological activity of ghrelin-desQ14 depends on the n-octanoyl group on serine 3. However, the greatest functional activity is achieved due to the ghrelin isoform of 28 a.a. (Hosoda et al. Biochem. Biophys. Res. Commun. 279 (3): 909-913 (2000)). Ghrelin is highly conserved, as human and rat ghrelin differ by only 2 amino acids (GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID No. 31) compared to GSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID No. 32)) (Kojima et al. 1999-6006. 40.

Ghrelin receptors (GHS-R) are expressed in various regions of the brain, including the arcuate nucleus (Arc) and the ventromedial nucleus of the hypothalamus and pituitary gland (Howard etal. Science 273: 974-977 (1996)); McKee et al. Mol Endokrin. 11: 415-423 (1997); Guan et al., Mol Brain Research 48: 23-29 (1997)), indicating that ghrelin predominantly acts in the brain. In addition to ghrelin stimulating GH release from the pituitary gland (Kojima et al. Nature 402: 656-660 (1999)), it has recently been identified as a key central regulator of nutrition (Nakazato et al., Nature 409: 194-198 (2001)). It has been specifically shown that after intracerebroventricular administration, ghrelin stimulates food intake. In addition, intracerebroventricular administration of anti-ghrelin antibodies inhibits food intake. Ghrelin injection induced increased NPY release, and anti-NPY antibodies together with AGRP antagonists blocked ghrelin-induced food intake, indicating that ghrelin modulates food intake by enhancing NPY and AGRP expression (Nakazato et al., Nature 409: 194-198 (2001 )). In addition, peripheral daily administration of ghrelin induced an increase in the body weight of mice and rats, and serum ghrelin concentrations increased in starving rats and decreased during feeding, further indicating that ghrelin plays a key role in regulating food intake (Tschop et al, Nature 407: 908-912). For transgenic rats expressing antisense to GHS-R RNA in Arc, less body weight and less adipose tissue are shown, which supports the view that ghrelin regulates body weight (Shuto et al., JCI 109: 14291436 (2002)) . There is also evidence that ghrelin plays a key role in people's eating behavior. Peripheral administration of ghrelin to humans increased appetite and increased food intake in humans (Wren et al., J Clin Endocrinol Metab 86: 5992-5998 (2001)). In people with Prader-Willi syndrome with the most common form of human syndromic obesity, extremely high levels of ghrelin are detected (Cummings et al. Nat Med 8: 643-644 (2002)). In addition, in humans, plasma ghrelin levels are greatly increased after diet-induced weight loss, correlating with rapid mass recovery after people stop the diet. In contrast, in patients with gastric bypass surgery, ghrelin levels remained low after diet, and in patients under these conditions their weight usually did not recover (Cummings et al., N Engl J Med 21: 1623-1630 (2002)). Therefore, ghrelin appears to be a key regulator of food intake and body weight in humans.

Since peripheral administration of ghrelin could increase food intake, leading to an increase in body weight (Tschop et al, Nature 407: 908-912), it is likely that ghrelin produced in the stomach reaches the brain through the bloodstream and switches eating behavior. Thus, it is possible that blocking the intake of ghrelin from the blood into the brain will stop the consumption of food in animals and humans. Since it has been shown that specific antibodies can block the action of ghrelin in the brain (Nakazato et al., Nature 409: 194-198 (2001)), it is likely that peripheral antibodies will also be able to block the action of peripheral ghrelin. In addition, since antibodies pass through the blood-brain barrier ineffectively, ghrelin-specific antibodies are likely to be able to isolate ghrelin from the brain, but will not act on ghrelin in the brain. This could be a particularly attractive opportunity, since ghrelin is also produced in the brain, where it probably performs different functions than regulating food intake (Nakazato et al., Nature 409: 194-198 (2001)). Therefore, a potential treatment for obesity could be the induction of ghrelin-specific antibodies in the host, leading to long-term blocking of ghrelin, leading to a decrease in food intake, similar to that observed in patients with bypass gastric anastomosis.

WO98 / 42840 describes the effect of ghrelin and fragments obtained from ghrelin on the gastrointestinal tract, and thus, in particular, their effects on gastric motility and gastric emptying. In addition, US 6,420,521 describes the use of short ghrelin peptides for affecting gastric function, including gastric emptying, gastric contractility, and glucose absorption.

However, it is generally difficult to induce an antibody responsive against peptides, especially against native peptides. One way to increase the effectiveness of vaccination is to increase the repeatability of the antigen used. Unlike individual proteins, viruses induce quick and effective immune responses in the absence of any adjuvants and with and without T cells (Bachmann and Zinkernagel, Ann. Rev. Immunol: 15: 235-270 (1991)). Although viruses often consist of few proteins, they are able to initiate significantly stronger immune responses than their individual components. For B-cell responses, it is known that one of the key factors for the immunogenicity of viruses is the repeatability and order of surface epitopes. Many viruses have a quasicrystalline surface that exhibits a regular set of epitopes that efficiently bind epitope-specific immunoglobulins on B cells (Bachmann and Zinkernagel, Immunol. Today 17: 553-558 (1996)). This binding of surface immunoglobulins to B cells is a strong activation signal that directly induces cell cycle progression and the production of IgM antibodies. Furthermore, such initiated B cells are capable of activating helper T cells, which in turn induce in the B cells switching of antibody production from IgM to IgG and the formation of long-lived memory B cells - the goal of any vaccination (Bachmann and Zinkernagel, Ann. Rev. Immunol 15: 235-270 (1997)). The structure of the virus is even associated with the formation of anti-antibodies in autoimmune disease and as part of the natural response to pathogens (see Fehr, T., et al., J Exp. Med. 185: 1785-1792 (1997)). Thus, antibodies represented by a highly organized viral surface are capable of inducing strong responses against antibodies.

However, as indicated, the immune system, as a rule, cannot form antibodies against its own structures. For soluble antigens presented in low concentrations, this is due to tolerance to Th-cell levels. Under these conditions, the binding of its own antigen to a carrier that is capable of providing T-cell assistance can impair tolerance. For soluble proteins present in high concentrations, or membrane proteins in low concentrations, B and Th cells may be tolerant. However, B-cell tolerance may be reversible (anergy) and can be destroyed by the administration of antigen in a highly organized form associated with a foreign carrier (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15: 235-270 (1997)).

SUMMARY OF THE INVENTION

The authors found that ghrelin or ghrelin peptides associated with a core particle having a structure with its own repeating organization and, in particular, for this case with virus-like particles (VLP) and VLP subunits, respectively, lead to highly organized and repeating conjugates, which are strong immunogens for induction of specific antibodies. The present invention relates to a prophylactic and therapeutic agent for treating obesity and related diseases based on an ordered and repetitive array of core particles with ghrelin or peptide derived from ghrelin, and in particular, on the conjugate and VLP-ghrelin / ghrelin peptide array, respectively. This prophylactic and therapeutic composition is capable of inducing high titers of antibodies against ghrelin in a vaccinated animal or human. Therefore, the present invention relates to ghrelin and its brain related properties. In addition, the present invention relates to the central effects of ghrelin on the brain, and more importantly, to the regulation of appetite, the secretion of growth hormone and energy homeostasis. Therefore, it is preferred that antibodies induced by the vaccination strategy of the present inventors are capable of binding to the n-octanoylated ghrelin form (s). As indicated, ghrelin or shorter peptide fragments of ghrelin can be used when they are bound to a core particle, and alternatively or preferably administered in adjuvant to induce ghrelin-specific antibodies in humans or animals.

Therefore, short peptide fragments of ghrelin, especially shorter peptides from residues 24-33, 42-51, 31-41, and 28-37, linked either at the C- or N-terminus to a core particle or virus-like particle, respectively, are capable of inducing highly specific antibodies against ghrelin, capable of neutralizing peripherally circulating ghrelin before it enters the central nervous system and affects growth hormone and, therefore, food intake.

Thus, in a preferred embodiment of the present invention, the antigen or antigenic determinant is selected from the group of ghrelin peptides corresponding to residues 24-33, 42-51, 31-41 and 28-37 of any of these sequences with SEQ ID No. 144 to 146, where these preferred ghrelin peptide fragments are selected from the group consisting of (a) human ghrelin; (b) bovine ghrelin; (c) sheep ghrelin; (d) canine ghrelin; (e) feline ghrelin; (f) mouse ghrelin; (g) pork ghrelin; and (h) equine ghrelin.

More specifically, in the present invention, the authors were able to induce high levels of antibodies that unexpectedly recognize the n-octanoylated form of ghrelin, as shown here, and in particular in example 17. In addition, the obtained antibodies also recognized an alternative isoform of ghrelin-desQ14. As a result, antibodies obtained by vaccination with ghrelin and ghrelin peptide, respectively, bound to the C- or N-terminus with a core particle or preferably VLP, were able to block the penetration of n-octanoylated ghrelin into the brain and modulated food intake in mice. Therefore, the present invention relates to vaccination strategies against active ghrelin, as a treatment for obesity and other related diseases.

As shown here, and in particular in Example 18, vaccination with ghrelin and ghrelin peptide, respectively, linked to the C- or N-terminus with a core particle, or preferably with VLP, leads to a decrease in mice consumption, suggesting that ghrelin and ghrelin peptide, respectively constitute a key regulator of food intake, and it is believed that antibodies directed to ghrelin and ghrelin peptide, respectively, are a possible therapeutic agent for obesity and other related diseases.

Thus, the present invention relates to a composition comprising: (a) a core particle with at least one first attachment site and (b) at least one antigen or antigenic determinant with at least one second attachment site, where said antigen or antigenic determinant is ghrelin or a peptide derived from ghrelin and wherein said second attachment site is selected from the group consisting of (i) an attachment site not found in nature with said antigen or antigenic child minant, and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site is capable of association with said first attachment site and where said antigen or antigenic determinant and said core particle interact through said association to form an ordered and a repeating array of antigens. Preferred embodiments of core particles suitable for use in the present invention are viruses, virus-like particles, bacteriophage, virus-like RNA phage particle, bacterial saw or flagellum, or any other core particle with its own repeating structure capable of forming an ordered and repeating array of antigens by the present invention.

More specifically, the invention relates to a composition comprising an ordered and repeating array of antigens or antigenic determinants and, in particular for this case, conjugates of ghrelin or peptide derived from ghrelin with VLP. More specifically, the invention relates to a composition comprising a virus-like particle and at least one ghrelin associated with it or a peptide derived from ghrelin. The invention also relates to a method for producing conjugates and ordered and repeating arrays, respectively. The compositions of the invention are suitable for the preparation of vaccines for the treatment of obesity and related diseases and as a pharmacine for the prevention or treatment of obesity and related diseases and for the effective induction of immune responses, especially antibody responses. In addition, the compositions of the invention are particularly suitable for efficiently inducing autospecific immune responses in this context.

In the present invention, ghrelin or a ghrelin-derived peptide is bound to a core particle and VLP, respectively, typically in an oriented manner, to produce an ordered and repeating array of ghrelin antigens or a ghrelin-derived peptide. In addition, the highly repetitive and organized structure of core particles and VLP, respectively, mediates the presentation of ghrelin or peptide derived from ghrelin in a highly ordered and repeating form, leading to a highly organized and repeating array of antigens. In addition, the binding of ghrelin or a peptide derived from ghrelin to a core particle and VLP, respectively, provides epitopes of helper T cells, since the core particle and VLP are foreign to the host immunized with the core particle ghrelin or the peptide obtained from ghrelin and an array of VLP ghrelin or a peptide derived from ghrelin, respectively. These arrays differ from conjugates previously known in the art, in particular, their highly organized structure, size and repeatability of antigen on the surface of the array.

In one aspect of the invention, ghrelin or a ghrelin-derived peptide is expressed in a suitable expression host or synthesized, while a core particle and VLP are respectively expressed and purified from an expression host suitable for laying and assembling a core particle and VLP, respectively. Ghrelin or ghrelin-derived peptides can be synthesized chemically. Since biologically active ghrelin includes n-octanoylated serine in the third position, chemical synthesis is the preferred way to obtain ghrelin for a vaccine containing biologically active forms of ghrelin. An array of ghrelin or ghrelin-derived peptide is then collected by binding ghrelin or ghrelin-derived peptide to a core particle or VLP, respectively.

In another aspect, the present invention relates to a composition comprising (a) a virus-like particle and (b) at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is ghrelin or a peptide derived from ghrelin and where at least , one specified antigen or antigenic determinant is associated with the specified virus-like particle.

In an additional aspect, the invention relates to a pharmaceutical composition comprising (a) the composition of the present invention and (b) an acceptable pharmaceutical carrier.

In a still further aspect, the present invention relates to a vaccine composition comprising a composition comprising (a) a core particle with at least one first attachment site and (b) at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is ghrelin or peptide derived from ghrelin and where said second attachment site is selected from the group consisting of (i) an attachment site that does not meet I am in nature with the indicated antigen or antigenic determinant, and (ii) an attachment site found in nature with the indicated antigen or antigenic determinant, where the specified second attachment site is capable of association with the specified first attachment site and where the specified antigen or antigenic determinant and said cow the particle interacts through this association to form an ordered and repeating array of antigens.

In an additional aspect, the present invention relates to a vaccine composition comprising a composition, wherein said composition comprises (a) a virus-like particle and (b) at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is ghrelin or derived from ghrelin a peptide and where at least one of the specified antigen or antigenic determinant is associated with the specified virus-like particle.

In yet a further aspect, the present invention relates to a method for producing a composition according to claim 1, comprising: (a) obtaining a virus-like particle and (b) obtaining at least one antigen and antigenic determinant, wherein said antigen or said the antigenic determinant is ghrelin or a peptide derived from ghrelin; (c) combining said virus-like particle and at least one said antigen or antigenic determinant such that at least one said antigen or antigenic determinant is associated with said virus-like particle.

Similarly, the present invention relates to a process for preparing a composition of the invention, comprising: (a) preparing a core particle with at least one first attachment site; (b) obtaining at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is ghrelin or a peptide derived from ghrelin, and wherein said second attachment site is selected from the group, consisting of (i) an attachment site not found in nature with the specified antigen or antigenic determinant, and (ii) an attachment site found in nature with the specified antigen or antigenic determinant, and where specified in Ora attachment site is capable of association to said first attachment site; and (c) combining said core particle and at least one said antigen or antigenic determinant, wherein said antigen or antigenic determinant and said core particle interact through said association to form an ordered and repeating array of antigens.

In another aspect, the present invention relates to a method of immunization, comprising administering the composition of claim 1 to an animal or human.

In an additional aspect, the present invention relates to the use of a composition according to claim 1 of the claims for the manufacture of a medicament for the treatment of obesity or related disease.

In yet a further aspect, the present invention relates to the use of a composition according to claim 1 for the manufacture of a medicament for the therapeutic or prophylactic treatment of obesity or related disease. In addition, in another further aspect, the present invention relates to the use of a composition according to claim 1, either alone or in combination with other agents for the preparation of a composition, vaccine or drug for the treatment or prevention of obesity or related disease and / or for stimulation mammalian immune system.

Therefore, the invention, in particular, relates to vaccine compositions suitable for the prevention and / or reduction or treatment of obesity or related conditions. The invention further relates to methods of immunization and vaccination, respectively, for preventing and / or reducing or treating obesity or related conditions in animals, and especially in domestic animals such as cats or dogs, as well as in humans. The compositions of the invention can be used prophylactically or therapeutically.

In particular implementations, the invention relates to methods for preventing, treating and / or reducing obesity or related conditions caused or enhanced by "own" gene products, i.e. “autoantigens,” as used herein. In related embodiments, the invention relates to methods for inducing immunological responses in animals and individuals, respectively, leading to the formation of antibodies, which prevents, treats and / or reduces obesity or related conditions caused or enhanced by “native” gene products.

As one of skill in the art can understand, when the compositions of the invention are administered to an animal or human, they may be in a composition containing salts, buffers, adjuvants or other substances desirable to increase the effectiveness of the composition. Examples of substances suitable for use in the preparation of pharmaceutical compositions are provided in numerous sources, including Remington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co. (1990)).

It is indicated that the compositions of the invention are “pharmacologically acceptable” if the individual receiving them can tolerate their administration. In addition, the compositions of the invention are administered in a “therapeutically effective amount” (ie, in an amount causing a desired physiological effect).

The compositions of the present invention can be administered by various methods known in the art, but are usually administered by injection, infusion, inhalation, oral administration, or other physical methods. The compositions can alternatively be administered intramuscularly, intravenously or subcutaneously. The components of the compositions for administration include sterile aqueous (e.g., saline) or non-aqueous solutions and suspensions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or an impermeable coating can be used to increase skin permeability and increase antigen absorption.

Other practitioners of the present invention will become apparent to those skilled in the art in light of what is known in the art, of the following description of the invention and claims.

BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

The figure 1 presents the products of the binding reaction of mouse C-ghrelin (SEQ ID: No. 77) or mouse ghrelin-GC (SEQ ID: No. 105) with the capsid protein Qβ. Lane M is a marker, lane 1 is a derivatized VLP of Qβ, lane 2 is Qβ-C-ghrelin in a soluble fraction, lane 3 is Qβ-C-ghrelin in an insoluble fraction, lane 4 is ghrelin-GC-Qβ in soluble fraction, lane 5 is ghrelin-GC-Qβ in the insoluble fraction. There is very little product in the insoluble fraction.

Figure 2 shows the average titers of ghrelin-specific IgG antibodies found in the total serum of mice immunized with mouse Qβ-C-ghrelin (Qb-cGhr), mouse ghrelin-GC-Qβ (Qb-GhrC) or PBS at 0, 14, 21 and 42 days. ELISA titers are presented as serum dilutions leading to half the maximum OD in an ELISA. ELISA plates were coated with mouse ghrelin with octanoylated serine (Bachem, Product No. H-4862) at a concentration of 20 μg / ml. The plates were blocked and then incubated with serially diluted murine sera from 14, 21, and 42 days. Bound antibodies were detected using an enzymatically labeled anti-mouse IgG antibody. As a control, serum from the same mice was also tested before immunization.

Figure 3 shows the total food intake of mice immunized with mouse Qβ-C-ghrelin (Qb-cGhr), mouse ghrelin-GC-Qβ (Qb-GhrC) or PBS. As a control, mice were immunized with PBS. All mice from 0 to 14 days were kept on a normal diet. Subsequently, all mice were fed a high fat diet (45%) to facilitate the development of diet-induced obesity. Food and water were given without restriction. At regular intervals after immunization (on the 5th, 11th, 14th, 21st, 28th, 35th, 40th, 49th, and 55th day after immunization), changes in body weight were monitored in each mouse, as well as the consumption of food and water per cell (i.e. group).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and substances similar or equivalent to those described herein can be used in the practice or for testing the present invention, the preferred methods and substances are described below.

1. Definitions:

Adjuvant: As used herein, the term “adjuvant” refers to non-specific stimulants of the immune response or substances that allow the host to form a depot that, when combined with the vaccine or pharmaceutical composition of the present invention, can accordingly provide an even stronger immune response. Many adjuvants may be used. Examples include Freund's complete and incomplete adjuvant, aluminum hydroxide, and a modified muramyl dipeptide. Additional adjuvants are mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronyl polyols, polyanions, peptides, oil emulsions, sea saucer hemocyanins, dinitrophenol, and potentially suitable human adjuvants such as BCG (Calmett-Guerin's bacillus) and Corynebacterium parvum . Such adjuvants are also well known in the art. Additional adjuvants that can be administered with the compositions of the present invention include, but are not limited to, a monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salts (alum), MF-59, OM-174, OM-197, OM-294 and virosomal adjuvant technology. Adjuvants may also contain a mixture of these substances.

Immunologically active saponin fractions obtained from the adjuvant, bark of the South American tree Quillaja Saponaria Molina, are known in the art. For example, QS21, also known as QA21, is an HPLC purified fraction from a Quillaja Saponaria Molina tree, and a process for its preparation (as QA21) is described in US Pat. No. 5,057,540. Also, adjuvants from Scott et al, Int. Archs. Allergy Appl. Immun., 1985, 77, 409 describe Kilaya saponins. Monophosphoryl lipid A and its derivatives are known in the art. A preferred derivative is 3-de-O-acylated monophosphoryl lipid A, and is known from UK Patent No. 2,220,211. Further preferred adjuvants are described in WO00 / 00462, the disclosure of which is incorporated herein by reference.

However, an advantageous feature of the present invention is the high immunogenicity of the compositions of the invention. As already indicated here and will become apparent throughout this specification, in additional alternative or preferred embodiments, vaccines and pharmaceutical compositions without adjuvants are provided, which leads to vaccines and pharmaceutical compositions for the treatment of AD without adjuvants and thus having a higher safety profile, how adjuvants can cause side effects. As used herein in the context of vaccines and pharmaceutical compositions for the treatment of AD, the term “without” refers to vaccines and pharmaceutical compositions used without adjuvants.

Amino acid linker: As used herein, an “amino acid linker,” or also simply referred to as a “linker” within this specification, either associates an antigen or antigenic determinant with a second attachment site, or more preferably already includes or contains a second attachment site, typically but not necessarily, as a single amino acid residue, preferably as a cysteine residue. However, as used herein, the term “amino acid linker” does not imply that such an amino acid linker consists solely of amino acid residues, even if an amino acid linker consisting of amino acid residues is a preferred embodiment of the present invention. Amino acid residues of the amino acid linker preferably consist of naturally occurring amino acids or non-natural amino acids known in the art, all L or all D, or mixtures thereof. However, the invention also includes an amino acid linker containing a molecule with a sulfhydryl group or a cysteine residue. Such a molecule preferably contains a C1-C6 alkyl, cycloalkyl (C5, C6), aryl or heteroaryl functional group. However, in addition to the amino acid linker, it is also necessary to include in the scope of the invention a linker, preferably containing a C1-C6 alkyl, cycloalkyl (C5, C6), aryl or heteroaryl functional group and not containing amino acids (s). The association between an antigen or antigenic determinant or, optionally, a second attachment site and an amino acid linker is preferably carried out via at least one covalent bond, more preferably at least one peptide bond.

Animal: As used here, the term "animal" is intended to include, for example, people, sheep, moose, deer, black-tailed deer, mink, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats , mice, birds, chickens, reptiles, fish, insects and arachnids.

Antibody: As used here, the term "antibody" refers to molecules capable of binding epitopes or antigenic determinants. The term is intended to include complete antibodies and their antigen binding fragments, including single chain antibodies. Most preferred antibodies are antigen binding fragments of human antibodies and include, but are not limited to, Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (scFv), single chain antibodies linked by Fvs disulfide bonds (sdFv), and fragments containing either V _L or V _H domain. Antibodies can come from any animal, including birds and mammals. Preferably, the antibodies are human, murine, rabbit, goat, guinea pig antibodies, camel, equine or chicken. As used here, the term "human" antibodies includes antibodies with the amino acid sequence of a human immunoglobulin and includes antibodies isolated from libraries of human immunoglobulins or from animals transgenic to one or more human immunoglobulins and not expressing endogenous immunoglobulins, as described, for example , in US patent No. 5939598 issued by Kucherlapati et al.

Antigen: As used here, the term "antigen" refers to a molecule that is capable of binding an antibody or T cell receptor (TCR), if it is represented by MHC molecules. As used herein, the term "antigen" also encompasses T-cell epitopes. The antigen is additionally capable of being recognized by the immune system and / or is able to induce a humoral immune response and / or cellular immune response, leading to activation of B and / or T lymphocytes. This, however, may require that, at least in some cases, the antigen contain or be associated with an Th cell epitope and that it is administered in an adjuvant. An antigen may have one or more epitopes (B and T epitopes). As suggested, the above specific reaction indicates that the antigen preferably reacts, as a rule, highly selectively with the corresponding antibody or TCR, and not with many antibodies or TCR induced by other antigens. As used herein, antigens may be mixtures of several individual antigens.

Antigenic determinant: As used here, the term "antigenic determinant" is intended to mean that part of the antigen that is specifically recognized by B or T lymphocytes. B-lymphocytes that respond to antigenic determinants form antibodies, while T-lymphocytes respond to antigenic determinants by proliferation and development of effector functions, which are important for mediating cellular and / or humoral immunity.

Association: As used here, the term "association", as applied to the first and second attachment sites, refers to the binding of the first and second attachment sites, preferably through at least one non-peptide bond. The bond may be covalent, ionic, hydrophobic, polar, or any combination thereof, preferably the type of bond is covalent.

Attachment site, first: As used herein, the phrase “first attachment site” refers to an element of non-natural or natural origin with which a second attachment site located on an antigen or antigenic determinant can be associated. The first attachment site may be a protein, polypeptide, amino acid, peptide sugar, polynucleotide, natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxygenin, metal ions, phenylmethyl sulfonyl fluoride), or a combination thereof, or chemically active groups thereof . The first attachment site is typically and preferably located on the surface of a core particle, preferably such as a virus-like particle. On the surface of the cow or virus-like particles, respectively, there are many first attachment sites, usually in a repeating configuration.

Attachment site, second: As used here, the phrase "second attachment site" refers to an element associated with an antigen or antigenic determinant with which a first attachment site located on the surface of a core particle or virus-like particle, respectively, can be associated. The second attachment site of the antigen or antigenic determinant may be a protein, polypeptide, peptide, sugar, polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxygenin, metal ions, phenylmethylsulfonyl fluoride), or a combination thereof, or their chemically active groups. At least one second attachment site is located on an antigen or antigenic determinant. Therefore, the term “antigen or antigenic determinant with at least one second attachment site” refers to an antigen or antigenic construct comprising at least an antigen or antigenic determinant and a second attachment site. However, in the antigen or antigenic determinant, in particular for the second attachment site of non-natural origin, i.e. not found in nature, this antigen or antigenic constructs contain an “amino acid linker”.

Communication: As used herein, the term “communication” refers to binding or attachment, which may be covalent, for example, by chemical bonding, or non-covalent, for example, ionic interactions, hydrophobic interactions, hydrogen bonds, and the like. Covalent bonds may, for example, be esters, ethers, phosphoesters, amides, peptides, imides, carbon disulfide bonds, phosphocarbon bonds, and the like. The term “connected” is broader than such terms as “connected”, “merged” and “attached”, and includes them.

Shell protein (s): As used herein, the term “shell protein (s)" refers to a bacteriophage or RNA phage protein (s) capable of being incorporated into the capsid construct of a bacteriophage or RNA phage. However, when referring to a particular gene product of an RNA phage coat protein gene, the term “CP” is used. For example, a particular gene product of the coat protein of the RNA phage Qβ coat is designated “CP Qβ”, while the “coat proteins” of the bacteriophage Qβ include “CP Qβ” as well as protein A1. The capsid of the bacteriophage Qβ mainly consists of CP Qβ with a small content of protein A1. Similarly, the VLP Qβ coat protein predominantly consists of CP Qβ with a small amount of A1 protein.

Crust particle: As used here, the term "crust particle" refers to a rigid structure with its own repeating structure. As used here, the core particle may be a product of a synthetic process or a product of a biological process.

Bound: As used herein, the term “bound” refers to attachment via covalent bonds or through strong non-covalent interactions, generally and preferably to attach via covalent bonds. Any method commonly used by those skilled in the art for binding biologically active substances can be used according to the invention.

Effective amount: As used herein, the term "effective amount" refers to the amount necessary or sufficient to realize the desired biological effect. An effective amount of a composition is the amount by which a given selected result is achieved, and such a quantity can be determined by one of ordinary skill in the art. For example, an effective amount for treating deficiency of the immune system may be the amount necessary to cause activation of the immune system, leading to the development of an antigen-specific immune response upon exposure of the antigen. The term is also synonymous with "enough."

The effective amount for any particular application may vary, depending on factors such as the disease or condition to be treated, the particular composition for administration, the size of the subject and / or the severity of the disease or condition. One skilled in the art, without the need for excessive experimentation, can empirically determine the effective amount of a particular composition of the present invention.

Epitope: As used here, the term "epitope" refers to continuous or discontinuous portions of a polypeptide with antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in humans. An epitope is recognized by an antibody or T cell through its T cell receptor, surrounded by an MHC molecule. As used herein, an “immunogenic epitope” is defined as part of a polypeptide that elicits an antibody response or induces a T-cell response in an animal, as determined by any method known in the art. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)). As used here, the term "antigenic epitope" is defined as part of a protein with which an antibody can immunospecifically bind its antigen, as defined by any method well known in the art. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not need to be immunogenic. Antigenic epitopes can also be T-cell epitopes, and in this case, they can immunospecifically bind to a T-cell receptor surrounded by an MHC molecule.

An epitope may include 3 amino acids in a spatial conformation unique to the epitope. Typically, an epitope consists of at least about 5 such amino acids, and more usually consists of at least about 8-10 such amino acids. If the epitope is an organic molecule, it can be as small as nitrophenyl.

Fusion: As used herein, the term "fusion" refers to combining amino acid sequences of different origins in a single polypeptide chain by combining within their reading frame their coding nucleotide sequences. The term "merger" in addition to merging with one of its ends explicitly includes internal mergers, i.e. inserts of sequences of a different origin in the polypeptide chain.

Ghrelin: As used herein, the term “ghrelin” refers to a peptide derived from a protein encoded by a ghrelin gene. As used herein, ghrelin includes all forms of ghrelin known in humans, cats, dogs and all domestic animals, as well as other animals. As used herein, ghrelin includes ghrelin with or without n-octanoyl modification. In addition, ghrelin also includes all the splicing options that exist in ghrelin. In addition, due to the high sequence homology in ghrelin of different species (only 2 a.a. in rats and humans) and human ghrelin (Kojima et al. Nature 402: 656-660 (1999)), all natural variants of ghrelin with more than 80% identity, preferably with more than 90%, more preferably with more than 95% and even more preferably with more than 99% with human ghrelin is referred to herein as “ghrelin”.

As used herein, the term “ghrelin-derived peptide” or “ghrelin peptides” is broadly defined as any peptide that is part of a ghrelin or contains at least two, preferably at least three, more preferably at least at least four, more preferably at least five or at least six and even more preferably at least eight or nine or even more preferably at least ten consecutive amino acids of the starting ghrelin peptide. Typically, the ghrelin peptide and the ghrelin fragment are used interchangeably. In addition, as used here, the terms "ghrelin peptide and its fragments" should include, in addition to the ghrelin peptide, any part of the specified ghrelin peptide, where the specified part can preferably be obtained by removing one or more amino acids from the N- and / or C-terminus. The ghrelin peptide can be obtained by recombinant expression in eukaryotic or prokaryotic expression systems, as a ghrelin peptide alone or as a hybrid with other amino acids or proteins, for example, to facilitate folding, expression or solubility of the ghrelin peptide or to facilitate the purification of the ghrelin peptide. To facilitate or enable proper folding of the hybrid proteins of ghrelin peptides and proteins of VLP subunits or capsids, one or more amino acids at the N- or C-terminus can be added to ghrelin peptides. At least one second attachment site can be added to the ghrelin peptide to allow the binding of ghrelin peptides to proteins of VLP subunits or capsids or core particles. Alternatively, ghrelin peptides can be synthesized using methods known in the art. Such peptides may even contain amino acids not contained in the corresponding ghrelin protein. Peptides can be modified by n-octanoylation.

Residue: As used herein, the term “residue” means a specific amino acid in a polypeptide backbone or side chain.

Immune response: As used herein, the term "immune response" refers to a humoral immune response and / or cellular immune response leading to the activation or proliferation of B and / or T lymphocytes and / or antigen presenting cells. However, in some cases, immune responses may be of low intensity and can only be detected by using at least one substance of the present invention. "Immunogenic" refers to an agent used to stimulate the immune system of a living organism so that one or more functions of the immune system are enhanced and directed against the immunogenic agent. An "immunogenic polypeptide" is a polypeptide that elicits a cellular and / or humoral immune response, either alone or when bound to a carrier in the presence or absence of an adjuvant. Preferably, the antigen-presenting cell may be activated.

A “enhancing” immune response substance is one in which, when a substance is added, an immune response that is more or more intense or, in any case, different when compared with the same immune response measured without adding a substance is observed. For example, the lytic activity of cytotoxic T cells can be measured, for example, using ⁵¹ Cr release analysis in samples obtained with or without a substance during immunization. It is believed that the amount of a substance at which the lytic activity of CTL increases when compared with the lytic activity of CTL without a substance is an amount sufficient to enhance the animal's immune response to the antigen. In a preferred embodiment, the immune response is increased at least about 2 times, more preferably about 3 times or more. You can also change the number or type of secreted cytokines. Alternatively, the amount of induced antibodies or their subclasses can also be changed.

Immunization: As used herein, the terms “immunize” or “immunization” or related terms refer to imparting the ability to exhibit a significant immune response (including antibodies and / or cellular immunity, such as effector CTLs) against a target antigen or epitope. These terms do not require that a complete immunity is created, but rather that an immune response is formed that is significantly larger than at the initial level. For example, a mammal can be considered immunized against a target antigen if, after applying the methods of the invention, a cellular and / or humoral immune response to the target antigen develops.

Natural origin: As used here, the term "natural origin" means that the whole or its part is not synthesized and exists or is obtained under natural conditions.

Unnatural: As used here, the term, as a rule, means not from nature, more specifically, the term means made by man.

Non-natural origin: As used here, the term "non-natural origin", as a rule, means synthetic or not from nature; more specifically, the term means made by man.

An ordered and repeating array of antigens or antigenic determinants: As used here, the term "ordered and repeating array of antigens or antigenic determinants" generally refers to a repeating structure of antigens or antigenic determinants, characterized by a typical and preferably uniform spatial arrangement of antigens or antigenic determinants with respect to to a core particle or virus-like particle, respectively. In one embodiment of the invention, the repeating structure may be a geometric structure. Typical and preferred examples of suitable ordered and repeating arrays of antigens or antigenic determinants are arrays having exactly repeating paracrystalline sequences of antigens or antigenic determinants, preferably in increments of 1 to 30 nanometers, preferably 5 to 15 nanometers.

Drank: As used herein, the term “drank” (the singular is “peel”) refers to the extracellular structures of bacterial cells composed of protein monomers (eg, pilin monomers) organized into ordered and repeating structures. In addition, pili represent structures involved in processes such as the attachment of a bacterial wall to the surface receptors of the host cell surface, intercellular genetic exchange, and cell-cell recognition. Examples of pili include Type 1, P-pili, F1C-pili, S-pili and 987P-pili. Additional examples of pili are discussed below.

Sawtooth structure: As used here, the phrase "sawtooth structure" refers to structures with similar characteristics to saws and consisting of protein monomers. One example of a “sawtooth structure” is a structure formed by a bacterial wall expressing modified pilin proteins that do not form ordered and repeating arrays that are identical to those of natural saws.

Polypeptide: As used here, the term "polypeptide" refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It means the molecular chain of amino acids and does not apply to a specific product length. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included in the definition of a polypeptide. This term is also intended to mean post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, etc. A recombinant or derivative polypeptide is not necessarily translated from the indicated nucleic acid sequence. It can also be obtained by any method, including chemical synthesis.

Autoantigen: As used herein, the term “autoantigen” refers to proteins encoded by the host DNA, and products formed by proteins or RNA encoded by the host DNA are defined as intrinsic. In addition, proteins formed by combining two or more of its own molecules or representing part of its own molecule and proteins having high homology with the second own molecule, as defined above (> 95%, preferably> 97%, more preferably> 99% ) can also be considered as your own.

Treatment: As used here, the terms "treatment", "treat", "treated" or "treating" refer to prevention and / or treatment. When used, for example, with respect to an infectious disease, the term refers to prophylactic treatment that increases the subject’s resistance to infection by a pathogen or, in other words, reduces the likelihood that the subject will become infected with the pathogen or show signs of the disease that are characteristic of the infection, as well as treatment after infection of the subject to fight the infection, for example, reducing or eliminating the infection or preventing deterioration. When used in relation to obesity or related diseases, the term "treatment" refers to prophylactic or therapeutic treatment that increases the subject's resistance to and / or reverses obesity.

Vaccine: As used herein, the term “vaccine” refers to a preparation containing a composition of the present invention and in a form that can be administered to an animal. Typically, the vaccine contains a traditional saline or buffered medium of an aqueous solution in which the composition of the present invention is suspended or dissolved. In this form, the composition of the present invention can easily be used to prevent, ameliorate, or otherwise treat a condition. After administration to a host, the vaccine is capable of provoking an immune response, including, but not limited to, the formation of antibodies and / or cytokines and / or activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and / or other types of cellular response.

Optionally, the vaccine of the present invention further includes an adjuvant, which may be present in a smaller or larger proportion with respect to the compound of the present invention.

Virus-like particle (VLP): As used here, the term "virus-like particle" refers to a structure similar to a virus particle. In addition, the virus-like particle of the present invention is non-replicable and non-infectious, since it lacks all or part of the viral genome, in particular the replicative and infectious components of the viral genome. The virus-like particle of the present invention may contain a nucleic acid different from its genome. A typical and preferred embodiment of the virus-like particle of the present invention is a viral capsid, such as a viral capsid of the corresponding virus, bacteriophage or RNA phage. As used hereinafter alternately, the terms “viral capsid” or “capsid” refer to a macromolecular structure consisting of subunits of a viral protein. Typically and preferably, the subunits of the viral protein are assembled into a viral capsid and capsid, respectively, having a structure with its own repetitive organization, where the specified structure, as a rule, is spherical or tubular. For example, capsid RNA phages or HBcAg have a spherical icosahedral symmetry. As used herein, the term “capsid-like structure” refers to a macromolecular structure consisting of subunits of a viral protein, similar to the capsid morphology in the sense defined above, but deviating from the usual symmetrical structure, albeit while maintaining a sufficient degree of orderliness and repeatability.

Virus-like particle of a bacteriophage: As used here, the term “virus-like particle of a bacteriophage” refers to a virus-like particle of a similar structure to a bacteriophage that is not replicating and infectious and that lacks at least the gene or genes encoding the bacteriophage replication apparatus and, as a rule, there is also no gene or genes encoding the protein or proteins responsible for the attachment of the virus or the penetration of the virus into the host. However, this definition should also encompass virus-like particles of bacteriophages in which the above gene or genes are still present but not active and therefore also lead to a non-replicating and non-infectious virus-like particle of the bacteriophage.

VLP or RNA phage coat protein: A capsid structure formed by self-assembly of 180 subunits of the RNA phage coat protein and optionally containing host RNA is referred to as “VLP or RNA phage coat protein”. A specific example is the VLP Qβ coat protein. In this particular case, the VLP Qβ coat protein can be assembled either exclusively from CP Qβ subunits (formed by expression of the CP Qβ gene containing, for example, the TAA stop codon that prevents any expression of the longer A1 protein due to suppression, see Kozlovska, TM, et al., Intervirology 39: 9-15 (1996)), or additionally comprise protein A1 subunit in the capsid structure.

Viral particle: As used here, the term "viral particle" refers to the morphological form of the virus. Some types of viruses contain a genome surrounded by a protein capsid; others have additional structures (e.g. shells, tails, etc.).

One or any: When the terms “one” or “any” are used in this description, they mean “at least one” or “one or more,” unless otherwise indicated.

As will be apparent to those skilled in the art, certain embodiments of the invention include the use of recombinant nucleic acid technologies such as cloning, polymerase chain reaction, DNA and RNA purification, expression of recombinant proteins in prokaryotic and eukaryotic cells, and the like. Such methods are well known to those skilled in the art and can be easily found in published laboratory method manuals (e.g., Sambrook, J. et al., Eds., Molecular Cloning, A Laboratory Manual, 2nd. Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel, F. et al., Eds., Current Protocols in Molecular Biology, John H. Wiley & Sons, Inc. (1997)). Fundamental laboratory technologies for working with tissue culture cell lines (Celis, J., ed., Cell Biology, Academic Press, 2nd edition, (1998)) and antibody-based technologies (Harlow, E. and Lane, D., Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988); Deutscher, MP, "Guide to Protein Purification", Meth. Enzymol. 128, Academic Press San Diego (1990); Scopes, RK, Protein Purification Principles and Practice, 3rd ed., Springer-Verlag, New York (1994)) are also adequately described in the literature, and all are incorporated herein by reference.

2. Compositions and methods for enhancing the immune response.

The disclosed invention relates to compositions and methods for enhancing the immune response against ghrelin or ghrelin peptide in an animal. The compositions of the invention comprise or, alternatively, consist of (a) a core particle with at least one first attachment site and (b) at least one antigen or antigenic determinant with at least one second attachment site, where said antigen or antigenic determinant is ghrelin or a peptide derived from ghrelin and wherein said second attachment site is selected from the group consisting of (i) an attachment site not found in nature with the specified antigen or antigenic determin antoi, and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site is capable of association with said first attachment site and where said antigen or antigenic determinant and said core particle interact through said association to form an ordered and a repeating array of antigens. More specifically, the compositions of the invention comprise or, alternatively, consist of a virus-like particle and at least one antigen or antigenic determinant, wherein the antigen or antigenic determinant is ghrelin or ghrelin peptide and where at least one antigen or antigenic determinant associated with a virus-like particle so as to form an ordered and repeating array of antigen-VLP. In addition, the invention allows practitioners to easily construct such a composition, in particular for the treatment and / or prophylactic prevention of obesity.

In one embodiment, the core particle comprises or is selected from the group consisting of a virus, a bacterial saw, a structure formed by a bacterial pilin, a bacteriophage, a virus-like particle, a virus-like RNA phage particle, a virus capsid particle, or a recombinant thereof. As the core particle of the invention, any virus known in the art with an ordered and repeating envelope and / or envelope protein structure can be selected; examples of suitable viruses include syndbis virus and other alphaviruses, rhabdoviruses (e.g. vesicular stomatitis virus), picornaviruses (e.g. human rhinovirus, Aichi virus), togaviruses (e.g. rubella virus), orthomyxoviruses (Togoto virus, Batken virus, bird virus plague), polyoma viruses (e.g., BK polyoma virus, JC polyoma virus, BFDV avian polyoma virus), parvoviruses, rotaviruses, Norfolk virus, foot and mouth disease virus, retrovirus, hepatitis B virus, tobacco mosaic virus, Sheep night virus and human papillomavirus pre revealing RNA phage, bacteriophage Qβ, bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophage NL95, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage MS2, bacteriophage f2, bacteriophage PP7 (for example, see Table 1 by Bachmann, MF and Zinkernagel , RM, Immunol. Today 17: 553-558 (1996)).

In a further embodiment, the invention utilizes genetic engineering of the virus to create a hybrid between the ordered and repeating viral envelope protein and the first attachment site containing a heterologous protein, peptide, antigenic determinant or active amino acid residue of choice, or, alternatively or preferably, being one. In constructing the compositions of the invention, other genetic manipulations known to those skilled in the art can be included; for example, it may be desirable to limit the replication ability of a recombinant virus through genetic mutation. In addition, the virus used for the present invention is replication defective due to chemical or physical inactivation or, as indicated, due to the lack of a replicable genome. The viral protein selected for fusion with the first attachment site must have an ordered and repeating structure. Such an organized and repeating structure includes paracrystalline structures in increments of 5-30 nm, preferably 5-15 nm, on the surface of the virus. Creating a hybrid protein of this type results in multiple, ordered and repetitive first attachment sites on the surface of the virus and reflects the normal structure of the natural viral protein. As will be appreciated by those skilled in the art, the first attachment site may or may not be part of any suitable protein, polypeptide, sugar, polynucleotide, peptide (amino acid), natural or synthetic polymer, secondary metabolite, or combinations thereof that may serve to specifically attach antigen or an antigenic determinant leading to an ordered and repeating array of antigens.

In another embodiment of the invention, the core particle is a recombinant alphavirus, and more specifically, a recombinant syndbis virus. Alphaviruses are positive chain RNA viruses that replicate their genomic RNA completely and without intermediate DNA in the cytoplasm of an infected cell (Strauss, J. and Strauss, E., Microbiol. Rev. 58: 491-562 (1994)). On some representatives of the alphavirus family, Sindbis (Xiong, C. et al., Science 243: 1188-1191 (1989); Schlesinger, S., Trends Biotechnol. 11: 18-22 (1993)), Semlica forest virus (SFV) (Liljestrom, P. & Garoff, H., Bio / Technology 9: 1356-1361 (1991)) and others (Davis, NL et al., Virology 171: 189-204 (1989)) have paid considerable attention to the application as virus-based expression vectors due to many different proteins (Lundstrom, K., Curr. Opin. Biotechnol. 8: 578-582 (1997); Liljestrom, P., Curr. Opin. Biotechnol. 5: 495-500 (1994)) and as candidates for vaccine development. Recently, a number of patents have been issued aimed at the use of alphaviruses for the expression of heterologous proteins and for the development of vaccines (see US Pat. Nos. 5,766,602; 5,792,462; 5,793,026; 5,789,245 and 5,814,482). The construction of the alphavirus core particles of the invention can be carried out by means generally known in the art of recombinant DNA technology, as described in the above articles, incorporated herein by reference.

To obtain virus-based core particles, many different recombinant host cells can be used to attach antigens or antigenic determinants. For example, it is known that alphaviruses have a wide range of hosts; Sindbis virus infects cultured mammalian, reptile and amphibian cells, as well as some insect cells (Clark, H., J. Natl. Cancer Inst. 51: 645 (1973); Leake, C., J. Gen. Virol. 35: 335 (1977); Stollar, V. in THE TOGAVIRUSES, RW Schlesinger, Ed., Academic Press, (1980), pp. 583-621). Thus, a plurality of recombinant host cells can be used in the practice of the invention. BHK, COS, Vero, HeLa, and CHO cells are particularly suitable for the preparation of heterologous proteins since they have the potential to glycosylate heterologous proteins in a manner similar to human cells (Watson, E. et al., Glycobiology 4: 227, (1994)) and they can be selected (Zang, M. et al., Bio / Technology 13: 389 (1995)) or genetically modified (Renner W. et al., Biotech. Bioeng 4: 476 (1995); Lee K. et al. Biotech. Bioeng 50: 336 (1996)) for growth in serum-free medium as well as in suspension.

The introduction of polynucleotide vectors into host cells can be carried out by methods described in standard laboratory manuals (see, for example, Sambrook, J. et al., Eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), chapter 9; Ausubel, F. et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997), chapter 16), including methods such as electroporation mediated by DEAE-dextran transfection, transfection, microinjection, cationic lipid-mediated transfection, transduction, scrape loading, ballistic administration and infection. Methods for introducing exogenous DNA sequences into host cells are discussed in Felgner, P. et al., US Pat. No. 5,580,859.

Packed RNA sequences can also be used to infect host cells. These packaged RNA sequences can be introduced into host cells by adding them to the culture medium. For example, the preparation of non-infectious alphavirus particles is described in a number of sources, including the Sindbis Expression System, Version C (Invitrogen Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for the production of virus-based core particles, these cells typically grow in tissue culture. Methods of growing cells in culture are well known in the art (see, for example, Celis, J., ed., CELL BIOLOGY, Academic Press, 2nd edition, (1998); Sambrook, J. et al., Eds., MOLECULAR CLONING , A LABORATORY MANUAL, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel, F. et al., Eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc. (1983)).

Additional examples of RNA viruses suitable for use as a core particle of the present invention include, but are not limited to, members of the Reoviridae family, including the genus Orthoreovirus (many serotypes of mammalian and avian retroviruses), the genus Orbivirus (catarrhal fever virus sheep, Eugenangee virus, Kemerovo virus, African horse disease virus and Colorado tick-borne fever virus), genus Rotavirus (human rotavirus, Nebraska calf diarrhea virus, mouse rotavirus, monkey rotavirus, cow or sheep rotavirus, avian rotavirus); Picomaviridae family, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, intestinal cytopathic human orphan (ECHO) viruses, hepatitis viruses A, C, D, E and G, monkey enteroviruses, mouse encephalomyelitis viruses (ME), mouse polio virus, bovine enteroviruses, swine enteroviruses), genus Cardiovirus (encephalomyocarditis virus (EMC), mengovirus), genus Rhinovirus (human rhinovirus, comprising at least 113 subtypes; other rhinoviruses), genus Apthovirus (FMDV (FMDV )); the Calciviridae family, including swine vesicular exanthema virus, San Miguel sea lion virus, Feline picornavirus and Norfolk virus; Togaviridae family, including the genus Alphavirus (eastern equine encephalitis virus, Semlica forest virus, Sindbis virus, chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Venezuelan equine encephalitis virus, western equine encephalitis virus), genus Flavirius mosquito-transmitted yellow fever virus, tropical fever virus, Japanese encephalitis virus, St. Lewis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunzhin virus, Central European tick-borne virus, Far Eastern transmitted mites virus Kyasanur Forest virus, Scottish Encephalitis virus, Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), the genus Pestivirus (mucosal disease virus, swine cholera virus, Border disease virus of sheep); the Bunyaviridae family, which includes the genus Bunyvirus (Bunyamvera and related viruses, viruses of the California encephalitis group), the genus Phlebovirus (Sicilian phlebotomic fever virus, Rift Valley fever virus), the genus Nairovirus (Congo-Crimean virus, Hemorrhiza hemorrhagic disease) and the genus Uukuvirus (Uukuniemi and related viruses); the Orthomyxoviridae family, including the genus Influenza virus (type A influenza virus, many human subtypes); swine and monkey influenza virus and equine influenza viruses; type B flu (many human subtypes) and type C flu (possibly a separate genus); the Paramyxoviridae family, which includes the genus Paramyxovirus (type 1 parainfluenza virus, Sendai virus, hemadsorption virus, type 2 to 5 parainfluenza viruses, Newcastle disease virus, mumps virus), the genus Morbillivirus (measles virus, subacute sclerosing panencephalitis virus, distemper virus, cattle plague virus), the genus Pneumovirus (respiratory syncytial virus (RSV), bovine respiratory syncytial virus and mouse pneumonia virus); the Rhabdoviridae family, including the genus Vesiculovirus (VSV), the Shandipur virus, the virus of the Flanders deer park), the genus Lyssavirus (rabies virus), rhabdoviruses of fish and filoviruses (Marburg virus and Ebola virus); the Arenaviridae family, including the lymphocytic choriomeningitis virus (LCM), the Tacaribe virus complex and the Lass virus; the Coronoaviridae family, including infectious bronchitis virus (IBV), mouse hepatitis virus, human intestinal coronavirus, and feline peritonitis virus (feline coronavirus).

Illustrative DNA viruses that can be used as core particles include, but are not limited to, the Poxviridae family, which includes the genus Orthopoxvirus (smallpox, alastrim, poxvaccine monkey, smallpox, buffalo smallpox, rabbit smallpox, ectromelia), genus Leporipoxvirus (myxoma, fibroma), genus Avipoxvirus (smallpox of birds, other avian poxviruses), genus Capripoxvirus (smallpox of sheep, smallpox of goats), genus Suipoxvirus (smallpox of pigs), genus Parapoxvirus (contagious pustular dermatitis virus, false bovine pox virus papular stomatitis but); family Iridoviridae (African swine fever virus, frog viruses 2 and 3, fish lymphocystosis virus); Herpesviridae family including alpha herpes viruses (herpes simplex types 1 and 2, chicken pox, equine abortion virus, herpes simplex virus 2 and 3, pseudorabies virus, infectious bovine keratoconjunctivitis virus, infectious bovine rhinotracheitis virus, feline rhinitis virus laryngotracheitis) beta herpes viruses (human cytomegalovirus and cytomegalovirus pigs, monkeys and rodents); gamma herpes viruses (Epstein-Bar virus (EBV), Marek's disease virus, Saimiri herpes, Ateles herpes virus, Sylvilagus herpes virus, guinea pig herpes virus, Lucke tumor virus); Adenoviridae family, including the genus Mastadenovirus (human subgroups A, B, C, D and E and ungrouped; monkey adenoviruses (at least 23 serotypes), infectious canine hepatitis and adenoviruses in cattle, pigs, sheep, frogs and many other species, the genus Aviadenovirus (avian adenoviruses) and uncultivated adenoviruses; the Papoviridae family, which includes the genus Papillomavirus (human papillomaviruses, bovine papillomaviruses, Shope rabbit papillomavirus and various pathogenic papillomaviruses of other species), Polyomav genus aquuolizing monkey virus (SV-40), vacuolating rabbit virus (RKV), K virus, BK virus, JC virus and other primate polyoma viruses such as lymphotropic papilloma virus); the Parvoviridae family includes the genus of adeno-associated viruses, the genus Parvovirus (feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc.) Finally, DNA viruses can include viruses such as chronic infectious neuropathic agents (CHINA virus).

In other implementations, bacterial pilin, a sub-fragment of a bacterial pilin, or a fusion protein containing either bacterial pilin or a sub-fragment thereof are suitably used to prepare the compositions and vaccine compositions of the invention. Examples of pilin proteins include pilins produced by Escherichia coli , Haemophilus influenzae , Neisseria meningitidis , Neisseria gonorrhoeae , Caulobacter crescentus , Pseudomonas stutzeri and Pseudomonas aeruginosa . Amino acid sequences of pilin proteins suitable for use in the present invention include those described in GenBank protocols AJ000636, AJ132364, AF229646, AF051814, AF051815 and X00981, the entire disclosures of which are incorporated herein by reference.

Bacterial pilin proteins, prior to the export of proteins to the bacterial periplasm, are typically processed to remove the N-terminal leader sequences. Further, as one of skill in the art can understand, the bacterial pilin proteins used for the compositions and vaccine compositions of the invention, respectively, as a rule, do not carry naturally occurring leader sequences.

One specific example of a pilin protein suitable for use in the present invention is E. coli P-pilin (GenBank protocol AF237482 (SEQ ID No. 1)). An example of an E. coli pilin of type 1 suitable for use in the present invention is a pilin with the amino acid sequence shown in the GenBank protocol P04128 (SEQ ID No. 2) encoded by the nucleic acid with the nucleotide sequence shown in the GenBank protocol M27603 (SEQ ID No. 3). Full descriptions of these GenBank protocols are incorporated herein by reference. In addition, the mature form of the above protein can generally be used to prepare the compositions and vaccine compositions of the invention, respectively.

Bacterial pilins or pilin sub-fragments suitable for use in the practice of the present invention are generally capable of being associated with the formation of ordered and repeating arrays of antigens.

Methods for producing pili and sawtooth structures in vitro are known in the art. For example, Bullitt et al., Proc. Natl. Acad. Sci. USA 93: 12890-12895 (1996) describe in vitro reduction of E. coli P-subunits. In addition, Eshdat et al., J. Bacteriol. 148: 308-314 (1981) describe methods suitable for dissociating E. coli type 1 pili and pili recovery. Briefly, these methods are as follows: pili dissociate by incubation at 37 ° C in saturated guanidine hydrochloride. The pilin proteins are then purified by chromatography, after which pilin dimers are formed by dialysis against 5 mM Tris (hydroxymethyl) aminomethanhydrochloride (pH 8.0). Eshdat et al. they also found that pilin dimers reassembled into pily form during dialysis against 5 mM Tris (hydroxymethyl) aminomethane (pH 8.0) containing 5 mM MgCl ₂ .

In addition, using, for example, traditional genetic engineering and protein modification methods, pilin proteins can be modified to contain a first attachment site, to which an antigen or antigenic determinant binds through a second attachment site. Alternatively, antigens and antigenic determinants through the second attachment site can be directly linked to amino acid residues present in these proteins by nature. These modified pilin proteins can then be used in the vaccine compositions of the invention.

The bacterial pilin proteins used to prepare the compositions and vaccine compositions of the invention, respectively, can be modified in a manner similar to the method described here for HBcAg. For example, cysteine and lysine residues can either be removed or replaced with other amino acid residues, and the first attachment sites can be added to these proteins. In addition, pilin proteins can either be expressed in a modified form or chemically modified after expression. Similarly, intact pills can be separated from bacteria and then chemically modified.

In another embodiment, pili or sawtooth structures are isolated from bacteria (e.g., E. coli ) and used to prepare the compositions and vaccine compositions of the invention. One example of pili suitable for preparing compositions and vaccine compositions is an E. coli type 1 pil, which is formed from pilin monomers with the amino acid sequence shown in SEQ ID No. 2.

A number of methods for isolating bacterial pili are known in the art. For example, Bullitt and Makowski (Biophys. J. 74: 623-632 (1998)) describe a method for cleaning pili to isolate P-pili from E. coli . In this method, the saws are cut off with hyperpiled E. coli containing the P-saw plasmid and purified by solubilization and precipitation cycles in MgCl ₂ (1.0 M).

After isolation, saws or sawtooth structures can be modified in a variety of ways. For example, a first attachment site can be added to the saws, to which antigens or antigenic determinants can be attached via the second attachment site. In other words, bacterial drank or sawtooth structures can be isolated and modified to obtain ordered and repeating arrays of antigens.

Antigens and antigenic determinants can be associated with natural cysteine or lysine residues located in the saw or sawtooth structures. In such cases, the high order and repeatability of the natural amino acid residue could direct the binding of antigens or antigenic determinants to saws or sawtooth structures. For example, drank or sawtooth structures can be linked to a second attachment site of antigens or antigenic determinants using a heterobifunctional crosslinking agent.

When structures that are synthesized by organisms in nature (e.g., drank) are used to prepare the compositions and vaccine compositions of the invention, it is often beneficial to genetically modify these organisms to produce structures that have the desired characteristics. For example, when E. coli type 1 drank is used, E. coli from which the drank is isolated can be modified to produce structures with specific characteristics. Examples of possible modifications to pilin proteins include the insertion of one or more lysine residues, the removal or replacement of one or more natural lysine residues, and the provision or replacement of one or more natural cysteine residues (for example, cysteine residues at positions 44 and 84 in SEQ ID No. 2) .

In addition, pilin genes can be subjected to additional modifications that result in the expression of products containing a first attachment site other than the lysine residue (for example, the FOS or JUN domain ). Of course, suitable first attachment sites are generally limited to those that do not prevent the pilin from forming pili or sawtooth structures suitable for use in the vaccine compositions of the invention.

Pilin genes found naturally in bacterial cells can be modified in vivo (for example, by homologous recombination), or pilin genes with specific characteristics can be introduced into these cells. For example, pilin genes can be introduced into bacterial cells as a component of either a replicating cloning vector, or a vector that integrates into a bacterial chromosome. The introduced pilin genes can also be linked to expression control control sequences (e.g., lac operator).

In most cases, the drank or sawtooth structures used in the compositions and vaccine compositions of the invention, respectively, consist of pilin subunits of the same type. Drank or sawtooth structures consisting of identical subunits are generally used, as they are expected to form structures that are highly ordered and repeating arrays of antigens.

However, the compositions of the invention also include compositions or vaccines containing pili or sawtooth structures formed by heterogeneous pilin subunits. The pilin subunits forming these drank or sawtooth structures can be expressed from genes found in a bacterial cell in nature, or can be introduced into cells. When the natural pilin gene and the introduced gene are both expressed in a cell forming pili or sawtooth structures, as a result, structures formed from a mixture of these pilin proteins are typically formed. In addition, when two or more pilin genes are expressed in a bacterial cell, the relative expression of each pilin gene is typically a factor determining the ratio of different pilin subunits to saw or sawtooth structures.

When drank or sawtooth structures having a specific composition of mixed pilin subunits are desired, the expression of at least one of the pilin genes can be regulated by a heterologous inducible promoter. Such promoters, as well as other genetic elements, can be used to regulate the relative amounts of various pilin subunits produced by a bacterial cell and, therefore, the composition of pili or sawtooth structures.

In addition, the antigen or antigenic determinant can be associated with bacterial saws or sawtooth structures through a bond that is not a peptide bond, bacterial cells producing saws or sawtooth structures used in the compositions of the invention can be genetically engineered to produce pilin proteins fused to the antigen or antigenic determinant. Such fusion proteins forming pili or sawtooth structures are suitable for use in the vaccine compositions of the invention.

Virus-like particles in the context of the present application relate to structures similar to a virus particle, but not pathogenic. In general, the virus-like particles lack the viral genome and therefore are non-infectious. Also, virus-like particles can be obtained in large quantities through heterologous expression, and they can be easily cleaned.

In a preferred embodiment, the core particle is a virus-like particle, wherein the virus-like particle is a recombinant virus-like particle. One of skill in the art can obtain VLP using recombinant DNA technology and virus coding sequences that are readily available publicly. For example, a virus or core protein envelope coding sequence can be constructed for expression in a baculovirus expression vector using a commercially available baculovirus vector under the regulatory control of a viral promoter with appropriate sequence modifications to allow functional binding of the coding sequence to the regulatory sequence. The coding sequence of the envelope of the virus or core protein can also be constructed for expression, for example, in a bacterial expression vector.

Examples of VLP include, but are not limited to, hepatitis B virus capsid proteins (Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus (Warnes, et al., Gene 160: 173-178 ( 1995)), Sindbis virus, Rotavirus (US 5071651 and US 5374426), foot and mouth disease virus (Twomey, et al., Vaccine 13: 1603-1610, (1995)), Norfolk virus (Jiang, X., et al., Science 250: 1580-1583 (1990); Matsui, SM, et al., J. Clin. Invest. 87: 1456-1461 (1991)), GAG retroviral protein (WO 96/30523), Ty p1 retrotransposon protein, surface protein hepatitis B virus (WO 92/11291), human papillomavirus (WO 98/15631), RNA phages, Ty, fr phage, GA phage, AP205 phage and Qβ phage.

As those skilled in the art will readily see, the VLP of the invention is not limited to any particular form. The particle can be synthesized chemically or through biological processes, which may be natural or unnatural. As an example, this type of implementation includes a virus-like particle or its recombinant forms.

In a more specific embodiment, the VLP may include or, alternatively, essentially consist of or, alternatively, consist of recombinant polypeptides or fragments thereof, selected from recombinant rotavirus polypeptides, recombinant Norfolk virus polypeptides, recombinant alphavirus polypeptides, recombinant recombinant viral polypeptides measles virus polypeptides, recombinant Sindbis virus polypeptides, recombinant polyoma virus polypeptides, recombinant retrovirus polypeptides, re recombinant hepatitis B virus polypeptides (e.g., HBcAg), recombinant tobacco mosaic virus polypeptides, recombinant sheep fox virus polypeptides, recombinant human papilloma virus polypeptides, recombinant bacteriophage polypeptides, recombinant polypeptide peptides, recombinant polypeptides, recombinant polypeptides, recombinant polypeptides, recombinant polypeptides phage GA and recombinant polypeptides of phage Qβ. A virus-like particle may further comprise or, alternatively, essentially consist of or, alternatively, consist of one or more fragments of such polypeptides, as well as variants of such polypeptides. Variants of the polypeptides may have, for example, at least 80%, 85%, 90%, 95%, 97% or 99% amino acid level identity with their wild-type copies.

In a preferred embodiment, the virus-like particle contains essentially consists of or alternatively consists of recombinant RNA phage proteins or fragments thereof. Preferably, the RNA phage is selected from the group consisting of a) bacteriophage Qβ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7; and m) bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-like particle contains or, alternatively, essentially consists of or alternatively consists of recombinant proteins of the RNA bacteriophage Qβ or RNA bacteriophage fr, or RNA bacteriophage AP205 or parts thereof.

In a further preferred embodiment of the present invention, recombinant proteins include, or, alternatively, essentially consist of, or, alternatively, consist of envelope proteins of RNA phages.

RNA phage coat proteins forming capsids or VLPs or bacteriophage coat protein fragments consistent with self-assembly into a capsid or VLP are therefore further preferred embodiments of the present invention. The envelope proteins of the bacteriophage Qβ, for example, can be recombinantly expressed in E. coli . Further, after such expression, these proteins spontaneously form capsids. Additionally, these capsids form a structure with their own repetitive organization.

Specific preferred examples of bacteriophage coat proteins that can be used to prepare compositions of the invention include coat proteins of RNA bacteriophages, such as the Qβ bacteriophage (SEQ ID No. 4; PIR Database, accession number VCBPQβ indicating CP Qβ and SEQ ID No. 5; accession number No. AAA16663 indicating A1 Qβ protein), bacteriophage R17 (SEQ ID No. 6; accession number PIR No. VCBPR7), bacteriophage fr (SEQ ID No. 7; accession number PIR No. VCBPFR), bacteriophage GA (SEQ ID No. 8; GenBank Part Number No. NP-040754), SP bacteriophage (SEQ ID No. 9; GenBank Part Number No. CAA30374, decrees identifying on SP CP and SEQ ID No. 10; Accession No. NP 695026, indicating SP protein A1), bacteriophage MS2 (SEQ ID No. 11; inventory number PIR No. VCBPM2), bacteriophage M11 (SEQ ID No. 12; GenBank inventory number AAC06250 ), bacteriophage MX1 (SEQ ID No. 13; GenBank accession number No. AAC14699), bacteriophage NL95 (SEQ ID No. 14; GenBank inventory number No. AAC14704), bacteriophage f2 (SEQ ID No. 15; GenBank Accession Number No. P03611), bacteriophage PP7 (SEQ ID No. 16) and bacteriophage AP205 (SEQ ID No. 28). In addition, Qβ protein A1 of bacteriophage Qβ (SEQ ID No. 5) or forms shortened at the C-terminus without 100, 150, or 180 amino acids at its C-terminus can be introduced into the capsid protein construct of the Qβ coat. Typically, the proportion of A1 Qβ protein relative to CP Qβ in the capsid construct is limited to ensure capsid formation.

It was also found that the Qβ coat protein is self-assembled into capsids when expressed in E. coli (Kozlovska TM. Et al., GENE 137: 133-137 (1993)). The resulting capsids or virus-like particles showed an icosahedral phage-like capsid structure with a diameter of 25 nm and a quasi-symmetry T = 3. In addition, the crystal structure of phage Qβ was resolved. The capsid contains 180 copies of the coat protein bound to the covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)), which leads to the amazing stability of the capsid from the Qβ coat protein. However, capsids or VLPs derived from a recombinant Qβ coat protein may contain subunits in the capsid that are not disulfide bonded to other subunits or partially bound. However, in VLPs, as a rule, approximately more than 80% of the subunits are linked to each other by disulfide bridges. Thus, after placing the recombinant Qβ capsid on the non-reducing SDS-PAGE, bands corresponding to the monomeric Qβ coat protein are visible, as well as bands corresponding to the hexameric or pentameric Qβ coat protein. Subunits partially linked by disulfide bonds on the non-reducing SDS-PAGE can be detected as a dimeric, trimeric or even tetrameric band. Qβ capsid protein also exhibits unusual resistance to organic solvents and denaturing agents. Unexpectedly, the inventors found that DMSO and acetonitrile in concentrations up to 30% and guanidine in concentrations up to 1 M do not affect the stability of the capsid. The high stability of the capsular protein of the Qβ coat is a beneficial feature, especially for its use in immunization and vaccination of mammals and humans of the present invention.

When expression in E. coli, the N-terminal methionine of the Qβ coat protein is typically removed, as observed by Edman N-terminal sequencing, as described by Stoll, E. et al. J. Biol. Chem. 252: 990-993 (1977). VLPs made up of Qβ shell proteins, where the N-terminal methionine is not removed, or VLPs including a mixture of Qβ shell proteins, where the N-terminal methionine is either cleaved or present, are also within the scope of the present invention.

Additional preferred virus-like particles of RNA phages, in particular Qβ, of the present invention are described in WO 02/056905, the description of which is fully incorporated herein by reference.

It was shown that additional envelope proteins of RNA phages also independently collect after expression in the bacterial host (Kastelein, RA. Et al., Gene 23: 245-254 (1983), Kozlovskaya, TM. Et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, MR. Et al., Virology 170: 238-242 (1989), Ni, CZ., Et al., Protein Sci. 5: 2485-2493 (1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). In addition to the envelope protein, the capsid of the phage Qβ capsid contains the so-called read to the end protein A1 and mature protein A2. A1 is formed by suppression in the UGA stop codon and has a length of 329 a.k. The capsid from the recombinant Qβ phage coat protein used according to the invention is devoid of A2 lysis protein and contains host RNA. The RNA phage coat protein is an RNA-binding protein and interacts with the stem loop of the ribosome-binding portion of the replicase gene, acting as a translational repressor during the life cycle of the virus. The sequence and structural elements of the interaction are known (Witherell, GW. & Uhlenbeck, OC. Biochemistry 28: 71-76 (1989); Lim F. et al., J Biol. Chem. 271: 31839-31845 (1996)). In general, it is known that the stem loop and RNA are involved in the assembly of the virus (Golmohammadi, R. et al., Structure 4: 543-5554 (1996)).

In a further preferred embodiment of the present invention, the virus-like particle contains, or, alternatively, essentially consists of, or, alternatively, consists of recombinant RNA phage proteins or fragments thereof, where the recombinant proteins include, or, alternatively, essentially consist of, or, alternatively, consist of mutant coat proteins of the RNA phage, preferably from mutant coat proteins of the above RNA phages. In another preferred embodiment, mutant RNA phage coat proteins are modified by removing at least one or, alternatively, at least two lysine residues by substitution, or by adding at least one lysine residue by substitution; alternatively, mutant RNA phage coat proteins are modified by removing at least one or, alternatively, at least two lysine residues, or by adding at least one lysine residue by insertion. Removing, replacing or adding at least one lysine residue allows you to vary the degree of binding, i.e. the number of Aβ1-6 peptides per subunit of VLP RNA phages, in particular, to meet and adapt to the requirements of the vaccine.

In another preferred embodiment, the virus-like particle comprises, or, alternatively, essentially consists of, or, alternatively, consists of recombinant proteins of the RNA bacteriophage Qβ or fragments thereof, where the recombinant proteins include, or, alternatively, essentially consist of, or alternatively consist of shell proteins with the amino acid sequence of SEQ ID No. 4 or a mixture of shell proteins with the amino acid sequences of SEQ ID No. 4 and SEQ ID No. 5 or mutant SEQ ID No. 5 and where the N-terminal methionine is preferably cleaved.

In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or, alternatively, consists of recombinant Qβ proteins or fragments thereof, where the recombinant proteins include, or, alternatively, essentially consist of, or, alternatively, consist of mutant Qβ coat proteins. In another preferred embodiment, these mutant envelope proteins are modified by removing at least one lysine residue by substitution or by adding at least one lysine residue by substitution. Alternatively, these mutant envelope proteins are modified by removing at least one lysine residue or by adding at least one lysine residue by insertion.

Four lysine residues are presented on the surface of the capsid from the Qβ coat protein. Mutant Qβ can also be used in the present invention for which the lysine residues shown are substituted with arginine residues. Thus, in the practice of the present invention, the following protein mutant Qβ coatings or mutant VLP Qβ coatings can be used: “Qβ-240” (Lys13-Arg; SEQ ID No. 17), “Qβ-243” (Asn10-Lys; SEQ ID No. 18), "Qβ-250" (Lys2-Arg, Lys13-Arg; SEQ ID No. 19), "Qβ-251" (SEQ ID No. 20) and "Qβ-259" (Lys2-Arg, Lys16-Arg; SEQ ID No. 21). Thus, in a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or, alternatively, consists of recombinant proteins of mutant Qβ coat proteins, including proteins with an amino acid sequence selected from group a) amino acid sequence SEQ ID No. 17; b) the amino acid sequence of SEQ ID No. 18; c) the amino acid sequence of SEQ ID No. 19; d) the amino acid sequence of SEQ ID No. 20; and e) the amino acid sequence of SEQ ID No. 21. The structure, expression and purification of the above Qβ, VLP envelope proteins and capsids from mutant Qβ coat proteins are respectively described in WO 02/056905. In particular, this applies to example 18 of the above application.

In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant Qβ proteins or fragments thereof, where the recombinant proteins include essentially consist of or, alternatively, consist of a mixture one of the previously mentioned mutant Qβ and the corresponding protein A1.

In a further preferred embodiment of the present invention, the virus-like particle contains, or alternatively essentially consists of, or, alternatively, consists of recombinant AP205 RNA phage proteins or fragments thereof.

The AP205 genome consists of a maturation protein, an envelope protein, a replicase, and two open reading frames that do not exist in related phages - the lysis gene and the open reading frame, which plays a role in the translation of the maturation gene (Klovins, J., et al., J. Gen. Virol . 83: 1523-33 (2002)). The AP205 envelope protein can be expressed from plasmid pAP283-58 (SEQ ID No. 27), which is a derivative of pQb10 (Kozlovska, TM et al., Gene 137: 133-37 (1993)) and contains the AP205 ribosome binding site. Alternatively, the AP205 coat protein can be cloned into pQb185, below the ribosome binding site present in the vector. Both approaches result in protein expression and capsid formation, as described in the co-pending US provisional patent application with the title "Molecular Antigenic Arrays" filed by the assignee of the present invention on July 17, 2002, fully incorporated herein by reference. The vectors pQb10 and pQb185 are vectors derived from the pGEM vector, and the expression of the cloned genes in these vectors is controlled by the trp promoter (Kozlovska, TM et al., Gene 137: 133-37 (1993)). Plasmid pAP283-58 (SEQ ID No. 27) contains the putative ribosomal binding region AP205 in the following sequence, which is located below the XbaI region and immediately above the start codon ATG of the AP205 envelope protein: tctagaATTTTCTGCGCACCCATCCCGGGTGGCGCCCAAA-GT GAGGAA A GAGGAA AGAGAA. Vector pQb185 contains the Shine-Dalgarno sequence below the XbaI region and above the start codeod (tctagaTTAACCCAACGCGT AGGAG TCAGGCCatg (SEQ ID No. 58), the Shine-Dalgarno sequence is underlined).

In a further preferred embodiment of the present invention, the virus-like particle contains, or alternatively essentially consists of, or, alternatively, consists of recombinant AP205 RNA phage coat proteins or fragments thereof.

Thus, this preferred embodiment of the present invention includes capsid-forming AP205 envelope proteins. Such proteins are expressed recombinantly or obtained from natural sources. AP205 envelope proteins produced in bacteria spontaneously form capsids, as evidenced by electron microscopy (EM) and immunodiffusion. The structural properties of the capsid formed by the AP205 envelope protein (SEQ ID No. 28) and the structural properties of the capsid formed by the AP205 RNA phage coat protein when viewed using EM are almost the same. AP205 VLPs are highly immunogenic and can be linked to antigens and / or antigenic determinants to obtain vaccine constructs representing antigens and / or antigenic determinants in a repetitive manner. High titers generated against the antigens presented in this way demonstrate that the associated antigens and / or antigenic determinants are available for interaction with antibody molecules and are immunogenic.

In a further preferred embodiment of the present invention, the virus-like particle contains, or alternatively essentially consists of, or, alternatively, consists of recombinant mutant AP205 RNA phage coat proteins or fragments thereof.

Assembly-suitable mutant forms of the VLP of AP205, including AP205 coat proteins with the substitution of Proline 5 for threonine at position 5 (SEQ ID NO: 29), can also be used to practice the present invention, and they lead to an additional preferred embodiment of the invention. VLP data, AP205 VLPs obtained from natural sources, or AP205 viral particles can be coupled with antigens to produce ordered repeating arrays of antigens of the present invention.

The mutant AP205 P5-T envelope protein can be expressed with pAP281-32 plasmid directly obtained from pQb185 (SEQ ID NO: 30) containing the AP205 mutant coat protein gene instead of the mutant Qβ coat protein gene. Vectors for expression of the AP205 envelope protein are transfected into E. coli for expression of the AP205 envelope protein.

Methods for expressing the coat protein and mutant coat protein, respectively, resulting in self assembly in the VLP are described in Examples 20 and 21. Suitable E. coli strains include, but are not limited to, E. coli K802, JM 109, RR1. Suitable vectors and strains and their combinations can be determined by testing the expression of the coat protein and mutant coat protein, respectively, by SDS-PAGE and the formation of the capsid and assembly by optionally first cleaning the capsids by gel filtration and then testing them in an immunodiffusion test (Ouchterlony test) or electron microscopy ( Kozlovska, TM et al., Gene 137: 133-37 (1993)).

The AP205 membrane proteins expressed from the vectors pAP283-58 and pAP281-32 may lack the initial amino acid methionine due to processing in the E. coli cytoplasm. Cleaved, unsplit forms of VLP AP205 or mixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or, alternatively, consists of a mixture of recombinant AP205 RNA phage coat proteins or fragments thereof and recombinant mutant AP205 RNA phage coat proteins or fragments thereof.

In a further preferred embodiment of the present invention, the virus-like particle comprises, or alternatively essentially consists of, or, alternatively, consists of fragments of recombinant coat proteins or recombinant mutant coat proteins of RNA phage AP205.

Fragments of recombinant AP205 coat proteins capable of being assembled into a VLP or capsid, respectively, are also useful in the practice of this invention. These fragments can be obtained by removing either the inside or ends of the coat protein and mutant coat protein, respectively. Insertions in the sequences of the coat protein and mutant coat protein, or fusion of antigenic sequences with sequences of the coat protein and mutant coat protein compatible with assembly in the VLP, are further embodiments of the invention and result in AP205 coat envelope proteins and particles, respectively. The result of insertions, deletions and fusions with the sequence of the envelope protein and whether it is compatible with the assembly in VLP can be determined by electron microscopy.

The particles formed by the AP205 sheath protein described above, sheath protein fragments, or hybrid sheath proteins can be isolated in pure form by combining the fractionation steps by precipitation and the purification steps by gel filtration using, for example, columns with CL-4B Sepharose, CL-2B Sepharose , Sepharose CL-6B and combinations thereof. Other methods for isolating virus-like particles are known in the art and can be used to isolate virus-like particles (VLPs) of the bacteriophage AP205. For example, US Pat. No. 4,918,166, incorporated by reference in its entirety, describes the use of ultracentrifugation to isolate Ty yeast retrotransposon VLP.

The crystal structure of some RNA bacteriophages has been determined (Golmohammadi, R. et al., Structure 4: 543-554 (1996)). Using this information, it is possible to identify residues present on the surface and, thus, it is possible to modify the envelope proteins of RNA phages so that by insertion or substitution it is possible to insert one or more chemically active amino acid residues. As a consequence, such modified forms of bacteriophage coat proteins can also be used for the present invention. Thus, variants of capsid-forming proteins or capsid-like structures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage AP205 and bacteriophage MS2) can also be used to prepare the compositions of the present invention.

Although the sequence of protein variants discussed above is different from their wild-type counterparts, these protein variants typically retain the ability to form capsids or capsid-like structures. Thus, the invention further includes compositions and vaccine compositions, respectively, further comprising protein variants forming capsids or capsid-like structures, as well as methods for preparing such compositions and vaccine compositions, respectively, of the individual protein subunits used to prepare such compositions and nucleic acid molecules acids encoding these protein subunits. Thus, variant forms of wild-type proteins that form capsids or capsid-like structures and retain the ability to associate and form capsids or capsid-like structures are included within the scope of the invention.

As a result, the invention further includes compositions and vaccine compositions, respectively, comprising proteins containing, or, alternatively, essentially consisting of, or, alternatively, consisting of amino acid sequences that are at least 80% 85 %, 90%, 95%, 97%, or 99% are identical to wild-type proteins that form ordered arrays and have their own repeating structure, respectively.

Additionally, nucleic acid molecules encoding the proteins used to prepare the compositions of the present invention are included within the scope of the invention.

In other implementations, the invention further includes compositions comprising proteins comprising, or, alternatively, essentially consisting of, or, alternatively, consisting of amino acid sequences of at least 80%, 85%, 90%, 95 %, 97% or 99% identical to any of the amino acid sequences shown in SEQ ID No: 4-21.

Proteins suitable for use in the present invention also include C-terminus-shortened protein mutants that form a capsid or capsid-like structure or VLP. Specific examples of such truncated mutants include proteins with the amino acid sequence shown in any of SEQ ID Nos: 4-21, where 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids. Typically, these C-terminated shortened mutants retain the ability to form capsids or capsid-like structures.

Additionally, proteins suitable for use in the present invention also include N-terminally shortened protein mutants that form capsids or capsid-like structures. Specific examples of such truncated mutants include proteins with the amino acid sequence shown in any of SEQ ID Nos: 4-21, where 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids. Typically, these N-terminally shortened mutants retain the ability to form capsids or capsid-like structures.

Additional proteins suitable for use in the present invention also include N- and C-terminus-shortened protein mutants that form capsids or capsid-like structures. Suitable truncated mutants include proteins with the amino acid sequence shown in any of SEQ ID Nos: 4-21, where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids are removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids are deleted from the C-terminus. Typically, data shortened at the N-terminus and C-terminus of the mutants retain the ability to form capsids or capsid-like structures.

The invention further includes proteins comprising, or, alternatively, essentially consisting of, or, alternatively, consisting of amino acid sequences of at least 80%, 85%, 90%, 95%, 97% or 99 % identical to the shortened mutants described above.

Thus, the invention includes compositions and vaccine compositions derived from capsid or VLP forming proteins, methods for producing these compositions from individual protein subunits and VLP or capsids, methods for producing data from individual protein subunits, nucleic acid molecules encoding these subunits, and methods for vaccinating and / or eliciting immunological responses in individuals using these compositions of the present invention.

In one embodiment, the invention relates to a vaccine composition of the invention further comprising an adjuvant. In another embodiment, there is no adjuvant in the vaccine composition. In a further embodiment, the vaccine composition comprises a core particle of the invention, wherein the core particle contains, and preferably is a virus-like particle, where preferably said virus-like particle is a recombinant virus-like particle. Preferably, the virus-like particle contains, or, alternatively, essentially consists of, or, alternatively, consists of recombinant RNA phage proteins or parts thereof, preferably of envelope proteins of RNA phages. In a preferred embodiment, the RNA phage coat protein has an amino acid sequence selected from the group consisting of: (a) SEQ ID No. 4; (b) a mixture of SEQ ID No. 4 and SEQ ID No. 5; (c) SEQ ID No. 6; (d) SEQ ID No. 7; (e) SEQ ID No. 8; (f) SEQ ID No. 9; (g) a mixture of SEQ ID No. 9 and SEQ ID No. 10; (h) SEQ ID No. 11; (i) SEQ ID No. 12; (k) SEQ ID No. 13; (1) SEQ ID No. 14; (m) SEQ ID No. 15; (n) SEQ ID No. 16 and (o) SEQ ID No. 28. Alternatively, recombinant proteins of the virus-like particle of the vaccine composition of the invention comprise, or, alternatively, essentially consist of, or, alternatively, consist of mutant RNA phage coat proteins, where said RNA phage is selected from the group consisting of (a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (k) bacteriophage f2; (1) bacteriophage PP7; and (m) bacteriophage AP205.

In a preferred embodiment, the mutant coat proteins of said RNA phage are modified by removal or by adding at least one lysine residue by substitution. In another preferred embodiment, the mutant coat proteins of said RNA phage are modified by removing at least one lysine residue or by adding at least one lysine residue by insertion. In a preferred embodiment, the virus-like particle comprises recombinant proteins of Qβ RNA phage, or alternatively, fr RNA phage, or AP205 RNA phage or fragments thereof.

As indicated previously, the invention includes virus-like particles or their recombinant forms. In one preferred embodiment, the particles used in the compositions of the invention comprise hepatitis B virus core protein (HBcAg) or an HBcAg fragment. In a further embodiment, the particles used in the compositions of the invention comprise hepatitis B virus core protein (HBcAg) or an HBcAg fragment modified either by the removal or reduction of a number of free cysteine residues. Zhou et al. (J. Virol. 66: 5393-5398 (1992)) have demonstrated that HBcAgs modified by the removal of natural cysteine residues retain the ability to associate and form capsids. Thus, VLPs suitable for use in the compositions of the invention include VLPs containing modified HBcAg or fragments thereof in which one or more naturally occurring cysteine residues are either removed or replaced by another amino acid residue (e.g., a serine residue).

HBcAg is a protein produced by processing the hepatitis B core antigen precursor protein. A number of HBcAg isotypes have been identified and their amino acid sequences are readily available to those skilled in the art. In most cases, the compositions and vaccine compositions of the invention are respectively prepared using a processed form of HBcAg (i.e., HBcAg from which the N-terminal leader sequence of the hepatitis B core antigen precursor protein has been removed).

In addition, when HBcAg is prepared under conditions where processing does not occur, HBcAg are typically expressed in a “processed” form. For example, when an E. coli expression system is used to produce the HBcAg of the invention, directing expression of the protein into the cytoplasm, these proteins are typically expressed such that the N-terminal leader sequence of the hepatitis B core antigen precursor protein is not provided.

The preparation of hepatitis B virus-like particles that can be used for the present invention is described, for example, in WO 00/32227, and therein, in particular, in Examples 17 to 19 and 21 to 24, as well as in WO 01/85208, and there, in particular, in examples 17 to 19, from 21 to 24, from 31 to 41, and in WO 02/056905. For the latter application, it is specifically referenced in Examples 23, 24, 31, and 51. All three documents are hereby incorporated by reference in their entirety.

The present invention also includes HBcAg variants modified by the removal or substitution of one or more additional cysteine residues. It is known in the art that free cysteine residues can be involved in a number of adverse chemical reactions. These adverse reactions include disulfide exchanges, reactions with chemicals or metabolites, which, for example, are administered or formulated when combined with other substances, or direct oxidation and reaction with nucleotides when exposed to UV light. Thus, toxic products can form, especially considering the fact that HBcAg has a strong tendency to bind nucleic acids. Thus, toxic products will be distributed between a variety of molecules that can individually be present in low concentrations, but reach toxic levels together.

In view of the above, one of the advantages of using HBcAg in vaccine compositions modified by the removal of natural cysteine residues is that the number of sites that toxic molecules can bind to when antigens or antigenic determinants are associated or are all removed is reduced.

A number of naturally occurring HBcAg variants have been identified that are useful in the practice of the present invention. For example, Yuan et al., (J. Virol. 73: 10122-10128 (1999)) describe variants in which the isoleucine residue at the position corresponding to position 97 in SEQ ID No. 22 is substituted with either a leucine residue or a phenylalanine residue. The amino acid sequences of a number of HBcAg variants, as well as some variants of the hepatitis B core antigen precursor variants, are described in the GenBank protocols AAF121240, AF121239, X85297, X02496, X85305, X85303, AF151735, X85259, X85286, X85260, X85317, X8585920, X85298, AF X85284 X85275 X72702 X85291 X65258 X85302 M32138 X85293 X85315 U95551 X85256 X85316 X85296 AB033559 X59795 X85299 , X85272, X85319, AB010289, X85285, AB010289, AF121242, M90520 (SEQ ID No. 24), P03153, AF110999 and M95589, the descriptions of each of which are incorporated herein by reference. The sequences of the above variants of the hepatitis B core antigen precursor are further described in WO 01/85208 in SEQ ID NO: 89-138. These HBcAg variants in the amino acid sequence differ in a number of positions, including amino acid residues corresponding to amino acid residues located at positions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID No. 25. Additionally, HBcAg variants that are suitable for use in the compositions of the present invention and which can additionally modify as described in this specification are described in WO 00/198333, WO 00/177158 and WO 00/214478.

As noted above, the processed or vaccine compositions of the present invention respectively employ, as a rule, processed HBcAg (i.e., those that lack leading sequences). The present invention includes vaccine compositions, as well as methods of using these compositions, in which the HBcAg variants described above are used.

Whether the amino acid sequence of a polypeptide has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to one of the above wild-type amino acid sequences or part thereof, can be arbitrarily determined using well-known computer programs such as Bestfit. When using Bestfit or another sequence alignment program to determine if a particular sequence, for example, has 95% identity with the reference amino acid sequence, the parameters are set so that the percentage of identity is calculated over the full length of the reference amino acid sequence and that homology gaps are allowed in the reference sequence up to 5% of the total number of amino acid residues.

The amino acid sequences of the above HBcAg variants and precursors are relatively similar to each other. Thus, a reference to the amino acid residue of an HBcAg variant located at a position corresponding to a specific position in SEQ ID No. 25 refers to the amino acid residue located at that position in the amino acid sequence shown in SEQ ID No. 25. Among hepatitis B viruses that infect mammals , the homology between these HBcAg variants is generally quite high, so that a person skilled in the art will not experience much difficulty considering both the amino acid sequence shown in SEQ ID No. 25 and amino acid sequence of a particular HBcAg, and identifying "corresponding" amino acid residues. In addition, the amino acid sequence of HBcAg shown in SEQ ID No. 24 showing the amino acid sequence of HBcAg obtained from the virus that infects the North American woodchuck has sufficient homology with the amino acid sequence of HBcAg shown in SEQ ID No. 25, so it is easy to understand that the three inserted amino acid residues are present in SEQ ID No. 23 between amino acid residues 155 and 156 of SEQ ID No. 25.

The invention also includes vaccine compositions containing HBcAg variants of hepatitis B viruses that infect birds, as well as vaccine compositions containing fragments of these HBcAg variants. For these HBcAg variants, one, two, three or more cysteine residues naturally occurring in these polypeptides can either be substituted with another amino acid residue or deleted before being incorporated into the vaccine compositions of the invention.

As discussed above, the removal of free cysteine residues reduces the number of sites where toxic components can bind to HBcAg, and also removes sites where crosslinking of lysine and cysteine residues of the same or neighboring HBcAg molecules can occur. Therefore, in another embodiment of the present invention, one or more cysteine residues of the hepatitis B virus capsid protein are either removed or replaced by another amino acid residue.

In other embodiments, the compositions and vaccine compositions of the invention respectively comprise HBcAg from which the C-terminal region has been removed (e.g., amino acid residues 145-185 or 150-185 from SEQ ID No. 25). Thus, further modified HBcAg suitable for use in the practice of the present invention include C-terminated shortened mutants. Suitable truncated mutants include HBcAg, where 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids are removed from the C-terminus.

Suitable for use in the practice of the present invention, HBcAg also include N-terminally shortened mutants. Suitable truncated mutants include modified HBcAg, where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids are removed from the N-terminus.

Additionally suitable for use in the practice of the present invention, HBcAg also include mutants truncated at the N- and C-terminus. Suitable truncated mutants include modified HBcAg, where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids are removed from the N-terminus and 1, 5, 10, 15, 20 are removed from the C-terminus , 25, 30, 34, 35 amino acids.

Additionally, the invention includes compositions and vaccine compositions, respectively, containing HBcAg polypeptides containing, or, alternatively, essentially consisting of, or, alternatively, consisting of amino acid sequences of at least 80%, 85%, 90%, 95 %, 97% or 99% identical to the shortened mutants described above.

In certain embodiments of the invention, a lysine residue is introduced into the HBcAg polypeptide to mediate the binding of ghrelin or ghrelin peptide to VLPs from HBcAg. In preferred embodiments, the compositions of the invention are prepared using HBcAg containing, or alternatively essentially consisting of, or, alternatively, consisting of amino acids 1-144 or 1-149, 1-185 of SEQ ID NO: 25, modified so that the corresponding at positions 79 and 80, the amino acids are replaced by a peptide with the amino acid sequence Gly-Gly-Lys-Gly-Gly (SEQ ID No. 33), which leads to the HBcAg polypeptide with the sequence shown in SEQ ID No. 26. In further preferred embodiments, cysteine residues at positions 48 and 107 in SEQ ID No. 25 for exchanged for serine. The invention further includes compositions containing the corresponding polypeptides, with the amino acid sequences shown in any of the above hepatitis B core antigen precursor variants that also possess the above amino acid changes. In addition, the scope of the invention includes additional variants of HBcAg capable of association with the formation of capsid or VLP and having the above amino acid changes. Thus, the invention further includes compositions and vaccine compositions, respectively, containing HBcAg polypeptides containing, or, alternatively, essentially consisting of, or, alternatively, consisting of amino acid sequences of at least 80%, 85%, 90 %, 95%, 97% or 99% identical to any of the wild-type amino acid sequences and the form of these proteins, which, where appropriate, are converted by removal of the N-terminal leader sequence, and where the above changes are made.

Compositions or vaccine compositions of the invention may contain mixtures of different HBcAg. Thus, these vaccine compositions may consist of HBcAg differing in amino acid sequence. For example, vaccine compositions containing wild-type HBcAg and modified HBcAg can be prepared in which one or more amino acid residues are altered (eg, deleted, inserted, or substituted). In addition, preferred vaccine compositions of the invention are vaccine compositions representing a highly ordered and repeating array of antigens, wherein the antigen is ghrelin or ghrelin peptide.

In a further preferred embodiment of the present invention, at least one ghrelin or ghrelin peptide is bound to said core particle and virus-like particle, respectively, by at least one covalent bond. Preferably, at least one ghrelin or ghrelin peptide is linked to a core particle or virus-like particle, at least one covalent bond where said covalent bond is a non-peptide bond leading to an ordered and repeating core particle ghrelin and the ghrelin array or conjugate - VLP respectively. This ghrelin-VLP array and conjugate, respectively, as a rule, and preferably has a repeating and ordered structure, since at least one, but usually more than one ghrelin or ghrelin peptide is associated with the VLP and core particle in an appropriately oriented manner. More than 120, preferably more than 180, preferably more than 270, and preferably more than 360 ghrelin or ghrelin peptides are preferably associated with VLPs. The formation of a repeating and oriented array and conjugate, respectively, of ghrelin - VLP and core particle, respectively, is provided by oriented and directed, as well as specific binding and attachment of at least one ghrelin or ghrelin peptide, respectively, to VLP and core particle, respectively, as will become clear in the future. In addition, the typical intrinsic highly repetitive and organized structure of VLP and core particles, respectively, predominantly promotes the presentation of ghrelin or ghrelin peptide in a highly oriented and repeating form, resulting in a highly organized and repeating ghrelin-VLP / core particle array or conjugate, respectively.

Therefore, the preferred conjugates and arrays according to the invention respectively differ from previous conjugates in this area in their highly organized structure, size and repeatability of antigen on the surface of the array. In addition, a preferred embodiment of the present invention allows the expression of both particles and antigen in the expression host, guaranteeing the correct folding of the antigen, i.e. at least one ghrelin or ghrelin peptide, and the proper folding and assembly of the VLP.

The present invention describes methods for binding ghrelin or ghrelin peptide to core particles and VLP, respectively. As indicated in one aspect of the invention, ghrelin or ghrelin peptide is coupled to a core particle and VLP, respectively, by crosslinking, typically and preferably using a heterobifunctional crosslinker. Several heterobifunctional crosslinking agents are known in the art. In preferred embodiments, heterobifunctional crosslinking agents comprise a functional group that can react with preferred first attachment sites, i.e. with the amino group of the side chain of the lysine residues of the core particle and the VLP or at least one VLP subunit, respectively, and an additional functional group that can react with a preferred second attachment site, i.e. a cysteine residue present in nature, made available for reaction by reduction, or incorporated into ghrelin or ghrelin peptide, and optionally also made available for reaction by reduction. The first stage of the procedure, usually called derivatization, is the reaction of a core particle or VLP with a crosslinking agent. The product of this reaction is an activated core particle or an activated VLP, also called an activated carrier. In a second step, unreacted crosslinking agent is removed using conventional methods, such as gel filtration or dialysis. In the third step, ghrelin or ghrelin peptide reacts with the activated carrier and this step is generally referred to as the binding step. Unreacted ghrelin or ghrelin peptide can optionally be removed in a fourth step, for example, by dialysis. Several heterobifunctional crosslinking agents are known in the art. These include preferred crosslinking agents SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other crosslinking agents available, for example, from Pierce Chemical Company (Rockford, IL, USA), with one functional group reacting with amino groups and one functional group reacting with cysteine residues. All of the above cross-linking agents after the reaction with the amino group lead to the formation of an amide bond, and after the reaction with cysteine, a thioether bond. Another class of crosslinking agents useful in the practice of the invention is characterized by the addition of a disulfide bond between ghrelin or ghrelin peptide and core particle or VLP upon binding. Preferred crosslinking agents belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). The degree of derivatization of the core particle and VLP, respectively, with a crosslinking agent can be influenced by changing experimental conditions, such as the concentration of each of the reacting partners, the excess of one reagent compared to the other, pH, temperature, and ionic strength. To meet vaccine requirements, the degree of binding, i.e. the amount of ghrelin or ghrelin peptide per core particle subunit and VLP, respectively, can be controlled by changing the experimental conditions described above. The solubility of ghrelin or ghrelin peptide may impose a limit on the amount of ghrelin or ghrelin peptide that can bind to each subunit and in cases where the resulting vaccine may be insoluble, reducing the amount of ghrelin or ghrelin peptide per subunit is beneficial.

A particularly preferred method for binding ghrelin or ghrelin peptide to a core particle and VLP, respectively, is the binding of a lysine residue on the surface of a core particle and VLP, respectively, to a cysteine residue on ghrelin or ghrelin peptide. Thus, in a preferred embodiment of the present invention, the first attachment site is a lysate residue, and the second attachment site is a cysteine residue. In some embodiments, binding to a core particle or VLP, respectively, may require the construction of a cysteine residue of an amino acid linker with ghrelin or ghrelin peptide containing as a second attachment site or part thereof. Alternatively, cysteine can be introduced either by insertion or mutation within a ghrelin or ghrelin peptide. Alternatively, a cysteine residue can be introduced via chemical coupling.

The choice of amino acid linker depends on the nature of the antigen and autoantigen, respectively, i.e. from the nature of ghrelin or ghrelin peptide, from its biochemical properties such as pI, charge distribution and glycosylation. Flexible amino acid linkers are generally preferred. Preferred embodiments of amino acid linkers are selected from the group consisting of (a) CGG; (b) an N-terminal gamma-1 linker; (c) an N-terminal gamma-3 linker; (d) Ig hinge regions; (e) N-terminal glycine linkers; (f) (G) _k C (G) _n with n = 0-12 and k = 0-5 (SEQ ID No. 34); (g) N-terminal glycine-serine linkers; (h) (G) _k C (G) _m (S) _i (GGGGS) _n with n = 0-3, k = 0-5, m = 0-10, l = 0-2 (SEQ ID No. 35) ; (i) GGC; (k) GGC-NH ₂ ; (1) C-terminal gamma-1 linker; (m) a C-terminal gamma-3 linker; (n) C-terminal glycine linkers; (o) (G) _n C (G) _k with n = 0-12 and k = 0-5 (SEQ ID No. 36); (p) C-terminal glycine-serine linkers; (q) (G) _m (S) _i (GGGGS) _n (G) _o C (G) _k with n = 0-3, k = 0-5, m = 0-10, l = 0-2 and o = 0-8 (SEQ ID No. 37).

Further preferred examples of amino acid linkers are the hinge region of immunoglobulins, glycine-serine linkers (GGGGS) _n (SEQ ID No. 38) and glycine linkers (G) _n , all further containing a cysteine residue as the second attachment site and, optionally, additional glycine residues. Typically, preferred examples of these amino acid linkers are N-terminal gamma-1: CGDKTHTSPP (SEQ ID No. 39); C-terminal gamma-1: DKTHTSPPCG (SEQ ID No. 40); N-terminal gamma-3: CGGPKPSTPPGSSGGAP (SEQ ID No. 41); Slotted gamma-3: PKPSTPPGSSGGAPGGCG (SEQ ID No. 42); N-terminal glycine linker: GCGGGG (SEQ ID No. 43); C-terminal glycine linker: GGGGCG (SEQ ID No. 44); C-terminal glycine-lysine linker: GGKKGC (SEQ ID No. 45); N-terminal glycine-lysine linker: CGKKGG (SEQ ID No. 46).

In a further preferred embodiment of the present invention, and especially if the antigen is a ghrelin peptide, GGCG (SEQ ID No. 47), GGC or GGC-NH ₂ ("NH ₂ " stands for amidation) at the C-terminus are preferred as amino acid linkers. peptide or CGG linkers at its N-terminus. Typically, glycine residues are inserted between bulk amino acids and cysteine for use as a second attachment site to avoid potential interference with a larger bulk amino acid in the binding reaction.

The cysteine residue present on ghrelin or ghrelin peptide must be in a reduced state to react with a heterobifunctional crosslinking agent on the activated VLP, i.e., free cysteine or a cysteine residue with a free sulfhydryl group must be available. For example, when the cysteine residue to function as an attachment site is in an oxidized form, for example, if it forms a disulfide bridge, it is necessary to restore this disulfide bridge, for example, DTT, TCEP or β-mercaptoethanol.

The binding of ghrelin or ghrelin peptide to the core particle and VLP, respectively, using a heterobifunctional crosslinking agent by the preferred methods described above provides oriented binding of ghrelin or ghrelin peptide to the core particle and VLP, respectively. Other methods for binding a ghrelin or ghrelin peptide to a core particle and VLP, respectively, include methods where the ghrelin or ghrelin peptide is crosslinked to the core particle and VLP, respectively, using carbodiimide-EDC and NHS. A ghrelin or ghrelin peptide can also be first modified with a thiol group by reaction, for example, with SATA, SATP or iminothiolan. Ghrelin or ghrelin peptide after deprotection, if necessary, can then be coupled to the core particle and VLP, respectively, as described below. After separation of the excess thioylating reagent, ghrelin and ghrelin peptide react with the core particle and VLP, respectively, previously activated heterobifunctional crosslinking agents containing a cysteine-reactive group and, therefore, representing at least one or more functional groups that react with cysteine residues with which thioylated ghrelin or ghrelin peptide can react as described above. Optionally, small amounts of a reducing agent are added to the reaction mixture. In additional methods, ghrelin or ghrelin peptide is coupled to the core particle and VLP, respectively, using a homobifunctional crosslinker such as glutaraldehyde, DSG, BM [PEO] ₄ , BS ³ (Pierce Chemical Company, Rockford, IL, USA), or other known homobifunctional crosslinkers with functional groups that react with amino groups or carboxyl groups on the core particle and VLP, respectively.

Other methods of binding VLP to ghrelin or ghrelin peptide include methods where the core particle and VLP are respectively biotinylated and ghrelin or ghrelin peptide are expressed as a streptavidin fusion protein, or methods where both ghrelin or ghrelin peptides and core particle and VLP are respectively biotinylated , for example, as described in WO 00/23955. In this case, ghrelin or ghrelin peptide can first be coupled to streptavidin or avidin by adjusting the ratio of ghrelin or ghrelin peptide to streptavidin so that the free attachment sites are still accessible for binding of the core particle and VLP, respectively, which are added in the next step. Alternatively, all components may be mixed in a "single vessel" reaction. Other ligand-receptor pairs can be used as binding agents for the binding of ghrelin or ghrelin peptide to the core particle and VLP, where a soluble form of the receptor and ligand is possible, which can cross-link with the core particle and VLP, respectively, and ghrelin or ghrelin peptide. Alternatively, either a ligand or a receptor can be fused to ghrelin or a ghrelin peptide, and thus mediate binding to the core particle and VLP, respectively, chemically bound or fused to either the receptor or the ligand, respectively. Hybridization can be accomplished by insertion or replacement.

As already indicated, in a preferred embodiment of the present invention, the VLP is a VLP of an RNA phage, and in an even more preferred embodiment, a VLP is a VLP from a Qβ RNA phage coat protein.

One or more antigen molecules can be associated with one subunit of the capsid or VLP of the envelope proteins of RNA phages, i.e. ghrelin or ghrelin peptide, preferably through unprotected lysine residues of VLP RNA phages, if sterically accessible. A specific feature of VLP from RNA phage coat proteins, and, in particular, VLP from Qβ coat protein, is therefore the ability to bind several antigens to one subunit. This allows the formation of dense arrays of antigens.

In a preferred embodiment of the invention, the binding and attachment of at least one ghrelin or ghrelin peptide to the core particle and virus-like particle, respectively, occurs through interaction and association, respectively, between at least one first attachment site of the virus-like particle and at least one a second attachment site of an antigen or antigenic determinant.

VLPs or capsids from Qβ coat proteins represent on their surface a certain amount of lysine residues with a certain topology with three lysine residues directed inside the capsid and interacting with RNA, and four other lysine residues exposed outward of the capsid. These specific properties help the binding of antigens to the outer surface of the particle, and not to the inner surface of the particle, where lysine residues interact with RNA. VLPs from the envelope proteins of other RNA phages also possess a certain amount of lysine residues on their surface and a certain topology of these lysine residues.

In further preferred embodiments of the present invention, the first attachment site is a lysine residue and / or the second attachment site contains a sulfhydryl group or a cysteine residue. In a very preferred embodiment of the present invention, the first attachment site is a lysine residue, and the second attachment site is a cysteine residue.

In a very preferred embodiment of the present invention, ghrelin or ghrelin peptide is linked by a naturally occurring ghrelin or peptide or designed cysteine residue to the lysine residues of the VLP of the RNA phage coat protein, and in particular to the VLP from the Qβ coat protein.

Another advantage of VLPs obtained from RNA phages is their high level of expression in bacteria, which ensures the formation of large quantities of material at an acceptable cost.

As indicated, the conjugates and arrays of the invention respectively differ from previous conjugates in this area in their highly organized structure, size and repeatability of antigen on the surface of the array. In addition, the use of VLP as carriers provides the formation of stable arrays and conjugates of antigens, respectively, with different antigenic densities. In particular, the use of VLP of RNA phages and, in particular, for this case, the use of VLP from the envelope proteins of the RNA phage Qβ provides a very high density of epitopes. The preparation of VLP compositions from envelope proteins of RNA phages with a high density of epitopes can be carried out using the instructions of this application. In a preferred embodiment of the invention, when the ghrelin peptide is preferably bound to VLP from Qβ coat proteins, the average amount of ghrelin peptides per subunit is preferred in an amount of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2 , 3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or more.

As defined here, the second attachment site may be present on an antigen or antigenic determinant in nature or may be unnatural.

In the absence of a suitable naturally occurring second attachment site on the antigen on the antigen or antigenic determinant, it is necessary to construct a non-natural second attachment site.

As described above, four lysine residues are exposed to the surface of the VLP from the Qβ coat protein. Typically, these residues in the reaction are derivatized with a crosslinking molecule. In the case where not all exposed lysine residues can bind to the antigen, the lysine residues that have reacted with the crosslinking agent remain with the crosslinking molecule bound to the ε-amino group after the derivatization step. This leads to the disappearance of one or more positive charges, which may be harmful to the solubility and stability of the VLP. By substituting some of the lysine residues for arginine residues, as in the Qβ coat protein mutants described below, the authors prevent the positive charges from disappearing excessively since the arginine residues do not react with a crosslinking agent. In addition, the replacement of lysine residues with arginine ones can lead to the formation of more specific arrays of antigens, since fewer sites are available for reaction with the antigen.

Thus, the exposed lysine residues are replaced by arginine in the following Qβ coat protein mutants and Qβ mutant VLPs presented in this application: Qβ-240 (Lys13-Arg; SEQ ID No. 17), Qβ-250 (Lys2-Arg, Lys13-Arg ; SEQ ID No. 19) and Qβ-259 (Lys2-Arg, Lys16-Arg; SEQ ID No. 21). The constructs are cloned, proteins are expressed, VLPs are purified and used to bind peptide or protein antigens. Also constructed are Qβ-251; (SEQ ID No. 20), guidance on how to express, purify, and bind VLP from the Qβ-251 coat protein can be found throughout this application.

In a further embodiment, the authors present a mutant Qβ coat protein with one additional lysine residue, suitable for producing even denser arrays of antigens. This mutant Qβ protein, Qβ-243 (Asn 10-Lys; SEQ ID No. 18) is cloned, the protein is expressed and isolated, and the capsid or VLP is purified, demonstrating that the introduction of an additional residue is compatible with self-assembly of subunits into a capsid or VLP. Thus, ghrelin or ghrelin peptide arrays and conjugates, respectively, can be obtained using VLP from mutant Qβ coat proteins. The practical method of binding antigens to VLP and, in particular, to VLP from RNA phage coat proteins is the binding of a lysine residue present on the VLP surface of RNA phage coat proteins to a cysteine residue that is naturally occurring or engineered on an antigen, i.e. e. ghrelin or ghrelin peptide. In order for the cysteine residue to be effective as a second attachment site, the sulfhydryl group must be available for binding. Thus, it is necessary that the cysteine residue is in a reduced state, i.e. a free cysteine or a cysteine residue with a free sulfhydryl group must be available. In the case where the cysteine residue for functioning as the second attachment site is in an oxidized form, for example, if it forms a disulfide bridge, it is necessary to restore this disulfide bridge, for example, DTT, TCEP or β-mercaptoethanol. For each antigen, it is necessary to adjust the concentration of the reducing agent and the molar excess of the reducing agent over the antigen. If necessary, starting from such low concentrations as 10 μM or less to concentrations of 10 to 20 mM or more, the titration range of the reducing agent is tested and antigen binding is determined. Although low concentrations of the reducing agent are compatible with the binding reaction, as described in WO 02/056905, higher concentrations inhibit the binding reaction, as is known to a person skilled in the art, and in this case, the reducing agent must be removed by dialysis or gel filtration. Advantageously, the pH of the dialysis or equilibrium buffer is lower than 7, preferably 6. The compatibility of the low pH buffer with antigen activity or stability is not tested.

The density of epitopes on VLP from surface proteins of RNA phages can be changed by choosing a crosslinking agent and other reaction conditions. For example, Sulfo-GMBS and SMPH crosslinkers typically achieve high epitope densities. Derivatization is positively influenced by a high concentration of reagents, and control of reaction conditions can be used to control the amount of VLP-bound RNA phage coat proteins, and especially VLP coat protein Qβ, of antigens.

Prior to the construction of the non-natural second attachment site, it is necessary to choose the position in which it needs to be merged, inserted or subjected to construction in a general sense. Thus, the position of the second attachment site must be chosen so as to avoid steric obstruction from the second attachment site or the amino acid linker containing it. In further embodiments, an antibody response directed to a site other than the site of interaction of the autoantigen with its natural ligand is desired. In such implementations, the second attachment site can be chosen so that it prevents the formation of antibodies against the site of interaction of the autoantigen with its natural ligands.

In most preferred embodiments, the ghrelin or ghrelin peptide comprises a second attachment site or a single reactive attachment site capable of association with the first attachment sites on the core particle and the VLP or VLP subunits, respectively. This provides a definite and uniform binding and association, respectively, of at least one, but usually more than one, preferably more than 10, 20, 40, 80, 120, 150, 180, 210, 240, 270, 300 , 360, 400, 450 antigens with core particles and VLP, respectively. Thus, providing a single second attachment site or a single reactive attachment site on an antigen guarantees a uniform and uniform type of binding and association, respectively, resulting in very highly ordered and repetitive arrays. For example, if binding and association are respectively effected by reacting lysine (as the first attachment site) and cysteine (as the second attachment site) according to the preferred embodiment of the invention, it is guaranteed that only one cysteine residue per antigen is present, whether present on the antigen the cysteine residue is natural or non-natural, capable of binding and association, respectively, with VLP and the first attachment site of the core particle, respectively.

In some implementations, the construction of a second attachment site on an antigen requires the fusion of an amino acid linker containing an amino acid suitable as a second attachment site according to the descriptions of the present invention. Therefore, in a preferred embodiment of the present invention, the amino acid linker is coupled to an antigen or antigenic determinant of at least one covalent bond. Preferably, the amino acid linker comprises a second attachment site or, alternatively, consists of it. In a further preferred embodiment, the amino acid linker contains a sulfhydryl group or a cysteine residue. In another preferred embodiment, the amino acid linker is cysteine. Some criteria for selecting an amino acid linker, as well as additional preferred embodiments of the amino acid linker according to the invention, are already indicated above.

In a further preferred embodiment of the invention, at least one antigen or antigenic determinant is fused respectively with a core particle and a virus-like particle, i.e. ghrelin or ghrelin peptide. As indicated above, a VLP typically consists of at least one subunit assembled in a VLP. Thus, in yet a further embodiment of the invention, the antigen or antigenic determinant is preferably at least one ghrelin or ghrelin peptide fused to at least one subunit of a virus-like particle or protein capable of being incorporated into the VLP to form a fusion protein of the VLP-subunit ghrelin or ghrelin peptide.

The fusion of ghrelin or ghrelin peptides can be carried out by inserting a VLP subunit in the sequence or by fusion with the N- or C-terminus of a VLP subunit or a protein capable of being incorporated into the VLP. Below, when referring to fusion proteins of a peptide with a VLP subunit, they mean fusion with either end of the subunit sequence or insertion of the peptide into the subunit sequence.

Fusion can also be carried out by inserting ghrelin or ghrelin peptide sequences into a variant of the VLP subunit, where a part of the subunit sequence is deleted, which is hereinafter referred to as shortened mutants. In truncated mutants, the N- or C-terminus or the internal parts of the VLP subunit sequence may be deleted. For example, a particular HBcAg VLP, for example, with the removal of amino acid residues 79 to 81, is a truncated mutant with an internal deletion. The fusion of ghrelin or ghrelin peptide or the N- or C-terminus of a truncated mutant of VLP subunits also leads to embodiments of the invention. Similarly, fusion of an epitope with a VLP subunit sequence can also be carried out by substitution, where, for example, for a particular HBcAg VLP, amino acids 79-81 are shaded by a foreign epitope. Thus, fusion, as described below, can be performed by inserting a ghrelin or ghrelin peptide sequence into a VLP subunit sequence by replacing part of the VLP subunit sequence with a ghrelin or ghrelin peptide sequence, or by a combination of deletion, replacement or insertions.

VLP subunit hybrid with ghrelin or ghrelin peptide is generally capable of self-assembly in VLP. VLPs representing epitopes fused to its subunits are also referred to herein as hybrid VLPs. As indicated, a virus-like particle contains or, alternatively, consists of at least one VLP subunit. In a further embodiment of the invention, the virus-like particle comprises or, alternatively, consists of a mixture of hybrid subunits and non-hybrid subunits, i.e. VLP subunits that do not carry the antigen fused with them, which leads to the so-called mosaic particles. This may be beneficial to ensure the formation and assembly of the VLP. In these embodiments, the ratio of the hybrid VLP subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or more.

Flanking amino acid residues can be added to either end of the sequence of the peptide or epitope for fusion with either the end of the sequence of the VLP subunit, or for internal insertion of such a peptide sequence into the VLP subunit sequence. Particularly preferred amino acids for use in flanking sequences added to ghrelin or ghrelin peptide for fusion are glycine and serine residues. Glycine residues provide additional flexibility, which can reduce the potentially destabilizing effect of the fusion of a foreign sequence with the sequence of the VLP subunit.

In a specific embodiment of the invention, the VLP is the VLP of hepatitis B core antigen. Hybrid proteins with either the N-terminus of HBcAg are described and are preferred embodiments (Neyrinck, S. et al., Nature Med. 5: 1157-1163 (1999)) or inserts in the so-called main immunodominant region (MIR) (Punpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), WO 01/98333). Also found are naturally occurring variants of HBcAgs with deletions in MIR (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001), specially incorporated by reference in their entirety), and fusions with the N- or C-terminus, and inserts at the MIR position corresponding to the deletion site, in comparison with wt HBcAg, are further embodiments of the invention. C-terminal fusions are also described (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)). A person skilled in the art will easily find guidance on how to construct hybrid proteins using classical molecular biology techniques (Sambrook, J. et al., Eds., Molecular Cloning, A Laboratory Manual, 2nd. Edition, Cold Spring Habor Laboratory Press, Cold Spring Harbor, NY (1989), Ho et al., Gene 77: 51 (1989)). Vectors and plasmids encoding HBcAg and HBcAg fusion proteins and suitable for expression of HBcAg and HBcAg fusion proteins are described (Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S. et al., Nature Med . 5: 1157-1163 (1999)), and they can be used in the practical implementation of the invention. Also by way of example (Example 6), the authors describe the insertion of an epitope in the HBIRAg MIR, resulting in a hybrid self-assembled HBcAg. An important factor for optimizing the efficiency of self-assembly and presentation of the epitope that will be inserted into the HBcAg MIR is the choice of the insertion site, as well as the number of amino acids that will be removed from the HBcAg sequence within the MIR (Pumpens, P. and Grens, E., Intervirology 44 : 98-114 (2001); EP 421635; US 6231864) upon insertion, or, in other words, which amino acids forming HBcAg are to be replaced by a new epitope. For example, amino acid substitution 76-80.79-81, 79-80, 75-85, or 80-81 for HBcAg is described by foreign epitopes (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001); EP0421635; US 6231864). HBcAg contains the long end of arginine residues (Pumpens, P. and Grens, E., Intervirology 44: 98-114 (2001)), which is not essential for capsid assembly and is capable of binding nucleic acids (Pumpens, P. and Grens, E. Intervirology 44: 98-114 (2001)). Containing this end of arginine residues or not containing HBcAg are embodiments of this invention.

In a further preferred embodiment of the invention, the VLP is the VLP of an RNA phage. The main envelope proteins of RNA phages expressed in bacteria and, in particular, in E. coli spontaneously assemble in VLP. Specific examples of bacteriophage coat proteins that can be used to prepare compositions of the invention include bacteriophage coat proteins such as bacteriophage Qβ (SEQ ID No. 4; PIR Database, Inventory No. VCBPQβ, indicating CP Qβ and SEQ ID No. 5 ; Inventory No. AAA16663 indicating A1 Qβ protein) and bacteriophage fr (SEQ ID No. 7; Inventory No. PIR VCBPFR).

In a more preferred embodiment, at least one ghrelin or ghrelin peptide is fused to a Qβ coat protein. Hybrid protein constructs are described where the epitopes are fused to the C-terminus of the truncated A1 Qβ protein form or inserted into A1 protein (Kozlovska, TM, et al., Intervirology, 39: 9-15 (1996)). Protein A1 is obtained by suppressing the stop codon of UGA, and it has a length of 329 a.k. or 328 a.k., if you take into account the cleavage of the N-terminal methionine. Cleavage of the N-terminal methionine in front of alanine (the second amino acid encoded by the CP Qβ gene) typically occurs in E. coli , and this is the case for the N-termini of the CP Qβ coat proteins. A portion of the A1 gene in the 3 ′ direction from the UGA amber codon encodes a CP extension of 195 amino acids. The insertion of at least one ghrelin or ghrelin peptide between positions 72 and 73 in the continuation of CP leads to further embodiments of the invention (Kozlovska, TM, et al., Intervirology 39: 9-15 (1996)). The fusion of ghrelin or ghrelin peptide with the C-terminus of the Qβ protein A1 shortened at the C-terminus leads to further embodiments of the invention. For example, Kozlovska et al., (Intervirology, 39: 9-15 (1996)) describe A1 Qβ fusion proteins, where the epitope is fused to the C-terminus of a CP Qβ extension shortened at position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)), the assembly of particles representing fusion epitopes for the formation of a mosaic particle typically requires the presence of a hybrid protein A1-ghrelin protein or peptide ghrelin, and wt CP. However, embodiments comprising virus-like particles and, in particular, for this case, VLP from the Qβ RNA phage coat protein, exclusively consisting of VLP subunits with at least one ghrelin or ghrelin peptide fused to them, are also in volume of the present invention.

The formation of mosaic particles can be carried out in a number of ways. Kozlovska et al., Intervirolog, 39: 9-15 (1996), describe two methods, both of which can be used in the practical implementation of the invention. In the first approach, the efficient presentation of a fusion epitope on VLP is mediated by expression of a plasmid encoding a A1 Qβ protein with a UGA stop codon between CP and a CP extension in an E. coli strain carrying a plasmid encoding a cloned suppressor for UGA tRNA, which leads to translation of the UGA codon into Trp (plasmid pISM3001 (Smiley BK, et al., Gene 134: 33-40 (1993))). In another approach, the stop codon of the CP gene is modified to UAA, the second plasmid expressing the A1-ghrelin protein hybrid or ghrelin peptide is transformed together. The second plasmid encodes different antibiotic resistance, and the beginning of replication is combined with the first plasmid (Kozlovska, TM, et al., Intervirology 39: 9-15 (1996)). In a third approach, the CP protein hybrid and the A1-ghrelin protein or ghrelin peptide are encoded as two cistrons functionally linked to a promoter such as a Trp promoter, as described in figure 1 in Kozlovska et al., Intervirology, 39: 9-15 ( 1996).

In a further embodiment, ghrelin or ghrelin peptide is inserted between amino acids 2 and 3 (numbering of cleaved CP, that is, where the N-terminal methionine is cleaved) CP fr, thus resulting in a ghrelin or ghrelin-CP fr peptide fusion protein. Vectors and expression systems have been described for constructing and expressing CP fr hybrid proteins that independently assemble in VLP and are useful in the practice of the invention (Pushko P. et al., Prot. Eng. 6: 883-891 (1993)). In a specific embodiment, the sequence of ghrelin or ghrelin peptide is inserted into the deletion variant CP fr after amino acid 2, where the residues CP fr 3 and 4 are removed (Pushko P. et al., Prot. Eng. 6: 883-891 (1993)).

Also described is the fusion of epitopes with the N-terminal protruding β-hairpin of the RNA phage coat protein MS-2 and the subsequent presentation of the fused epitope on the self-assembled VLP RNA phage MS-2 (WO 92/13081), and the fusion of ghrelin or ghrelin peptide by insertion or substitutions in the envelope protein of the RNA phage MS-2 also fall within the scope of the invention.

In another embodiment, ghrelin or ghrelin peptides are fused to a papilloma virus capsid protein. In a more specific embodiment, ghrelin or ghrelin peptides are fused to the main protein of the capsid L1 of the cow virus papilloma virus type 1 (BPV-1). Vectors and expression systems have been described for the construction and expression of BPV-1 fusion proteins in baculovirus / insect cell systems (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00 / 23955). Replacing amino acids 130-136 L1 BPV-1 with ghrelin or ghrelin peptide results in a hybrid protein L1 BPV-1-ghrelin or ghrelin peptide, which is a preferred embodiment of the invention. Cloning in the baculovirus vector and expression in baculovirus-infected Sf9 cells are described, and can be used in the practice of the invention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373-2378 (1999), WO 00 / 23955). Purification of collected particles representing ghrelin fusion or ghrelin peptides can be carried out in a number of ways, for example, by gel filtration or sucrose gradient ultracentrifugation (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96: 2373- 2378 (1999), WO 00/23955).

In a further embodiment of the invention, ghrelin or ghrelin peptides are fused to a Ty protein capable of being incorporated into Ty VLP. In a more specific embodiment, ghrelin or ghrelin peptides are fused to p1 or a capsid protein encoded by the TYA gene (Roth, JF, Yeast 16: 785-795 (2000)). The yeast retrotransposons Ty1, 2, 3, and 4 are isolated from Saccharomyces cerevisiae , while the retrotransposon Tf1 is isolated from Schizosaccharomyces Pombae (Boeke, JD and Sandmeyer, SB, "Yeast Transposable elements", in The molecular and Cellular Biology of the Yeast Genome , Protein Synthesis, and Energetics., P. 193, Cold Spring Harbor Laboratory Press (1991)). Retrotransposons Ty1 and 2 belong to the class of copia elements of plants and animals, while Ty3 belongs to the gypsy family of retrotransposons belonging to plant and animal retroviruses. In the Ty1 retrotransposon, the p1 protein, also referred to as Gag or capsid protein, has a length of 440 amino acids. P1 during VLP maturation is cleaved at position 408, which leads to p2 protein, an integral component of VLP.

Hybrid proteins with p1 and vectors for expressing said hybrid proteins in yeast are described (Adams, SE, et al., Nature 329: 68-70 (1987)). For example, ghrelin or ghrelin peptide can be fused to p1 by inserting a sequence encoding ghrelin or ghrelin peptide into the BamH1 region of pMA5620 plasmid (Adams, SE, et al., Nature 329: 68-70 (1987)). Cloning of sequences encoding foreign epitopes into the pMA5620 vector results in the expression of fusion proteins containing amino acids 1-381 p1 Ty1-15, with the C-terminus fused to the N-terminus of the foreign epitope. Similarly, the N-terminal fusion of ghrelin or ghrelin peptides, or an internal insertion into the p1 sequence, or the replacement of part of the p1 sequence is also intended to be within the scope of the invention. In particular, the insertion of ghrelin or ghrelin peptides into the Ty sequence between amino acids of the p1 Ty protein 30-31, 67-68,113-114 and 132-133 (EP0677111) leads to preferred embodiments of the invention.

Additional VLPs suitable for fusion with ghrelin or ghrelin peptides are, for example, retrovirus-like particles (WO 9630523), HIV Gag (Kang, YC, et al, Biol. Chem. 380: 353-364 (1999)), virus cow pea mosaics (Taylor, KM et al., Biol. Chem. 380: 387-392 (1999)), VLP of parvovirus VP2 (Rueda, P. et al., Virology 263: 89-99 (1999)), HBsAg ( US 4722840, EP0020416B1).

Examples of hybrid VLPs suitable for practicing the invention are also the VLPs described in Intervirology 39: 1 (1996). Additional examples of VLP contemplated for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, tobacco virus mosaics. Virus-like particles of SV-40, polyoma virus, adenovirus, herpes simplex virus, rotavirus and Norfolk virus have also been constructed, and hybrid VLPs of these VLPs are also within the scope of the present invention.

In a further preferred embodiment of the present invention, the antigen or antigenic determinant is ghrelin or ghrelin peptide.

In a further highly preferred embodiment of the invention, the antigen or antigenic determinant is selected from the group consisting of a) human ghrelin; b) feline ghrelin; c) canine ghrelin; d) bovine ghrelin; e) sheep ghrelin; f) equine ghrelin; g) pork ghrelin; h) a peptide or fragment thereof of any ghrelin from a) to g).

In a further highly preferred embodiment of the invention, the antigen or antigenic determinant comprises, or, alternatively, essentially consists of, or, alternatively, consists of an amino acid sequence selected from the group consisting of:

(a) GSSFLSPEHQRVQRKESKKPPAKLQPR (SEQ ID No. 48)

(b) GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID No. 31)

(c) GSSFLSPEHQKLQQRKESKKPPAKLQPR (SEQ ID No. 49)

(d) GSSFLSPEHQKLQRKESKKPPAKLQPR (SEQ ID No. 50)

(e) GSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID No. 32)

(f) GSSFLSPEHQKAQRKESKKPPAKLQPR (SEQ ID No. 51)

(g) KKPPAKLQPR (SEQ ID No. 52)

(h) PPAKLQPR (SEQ ID No. 53)

(i) AKLQPR (SEQ ID No. 54)

(j) GSSFLSPEHQ (SEQ ID No. 55)

(k) EHQRVQQRKE (SEQ ID No. 56)

(l) KLQPR (SEQ ID No. 59)

(m) GSSFLSPEHQRVQ (SEQ ID No. 60)

(n) QRKESKKPPAKLQPR (SEQ ID No. 61)

(o) GSSFLSPEHQKLQ (SEQ ID No. 62)

(p) QRKESKKPPAKLQPR (SEQ ID No. 63)

(q) EHQRVQQRKES (SEQ ID No. 111)

(r) EHQKAQQRKE (SEQ ID No. 112)

(s) EHQKAQQRKES (SEQ ID No. 113)

(t) EHQKLQQRKE (SEQ ID No. 114)

(u) EHQKLQQRKES (SEQ ID No. 115)

(v) LSPEHQRVQQ (SEQ ID No. 116)

(w) LSPEHQKAQQ (SEQ ID No. 117)

(x) LSPEHQKLQQ (SEQ ID No. 118).

In a further highly preferred embodiment of the invention, the antigen or antigenic determinant is human, canine, pork, equine, sheep, bovine, guinea pig, canine or mouse ghrelin and ghrelin peptide, respectively. Ghrelin and ghrelin peptides, respectively, can be obtained by expression of DNA encoding ghrelin and ghrelin peptide, respectively, under the control of a strong promoter. Preferably, the DNA is encoded not by the precursor of progrelin, but only by the peptide framework of the active n-octanoyl-modified peptide. Various examples of this are described in the literature, and they can be used, possibly after modifications, for the expression of ghrelin of any desired species.

In a further preferred embodiment of the invention, the antigen or antigenic determinant is a ghrelin-derived peptide or fragment thereof. Preferably, the ghrelin peptide is selected from the group consisting of a) a human ghrelin peptide; b) bovine ghrelin peptide; c) a feline ghrelin peptide; d) canine ghrelin peptide; e) porcine ghrelin peptide; f) chicken ghrelin peptide; g) mouse ghrelin peptide; h) equine ghrelin peptide; i) sheep ghrelin peptide; and j) a fragment of any ghrelin peptide from a) -i).

Such ghrelin peptides or fragments thereof can be obtained using standard molecular biology methods, where the nucleotide sequence encoding the fragment of interest is amplified by GWH and cloned as a fusion with a polypeptide tag, such as a histidine tag, Flag, myc, or the constant region of an antibody (region Fc). By introducing the enterokinase cleavage site between the ghrelin fragment and the label, the ghrelin fragment, after purification, can be separated from the label by cleavage with enterokinase. In another approach, a ghrelin fragment can be synthesized in vitro with or without n-octanoyl modification using standard peptide synthesis reactions known to one skilled in the art. In an additional approach, fragments of ghrelin can be obtained by cleavage with a protease or chemical cleavage of full-sized ghrelin, where both methods are well known to specialists in this field.

In a further preferred embodiment of the present invention, the ghrelin peptide comprises at least one antigenic site of ghrelin. One of skill in the art knows how to identify the corresponding peptides and amino acid sequences, respectively. Preferably, the fragments may include the N- or C-terminus of ghrelin. The C-terminus may be particularly suitable since n-octanoyl modification, which may possibly inhibit the binding of the unmodified form specific antibodies, is near the N-terminus.

Preferred human ghrelin peptides include:

KKPPAKLQPR (SEQ ID No. 52)

PPAKLQPR (SEQ ID No. 53)

AKLQPR (SEQ ID No. 54)

KLQPR (SEQ ID No. 59)

GSSFLSPEHQ (SEQ ID No. 55)

EHQRVQQRKES (SEQ ID No. 111)

LSPEHQRVQQ (SEQ ID No. 116)

GSSFLSP (SEQ ID No. 119)

However, peptides from internal ghrelin sites may also be attractive candidates for vaccines. Since ghrels of various species are highly homologous, it is likely that responses can be induced by cross-reactive antibodies. Thus, an antibody that responds to canine or mouse ghrelin also recognizes human ghrelin and vice versa. Therefore, it is within the scope of this invention that all ghrelin and peptides derived from them with amino acids of more than 80%, preferably more than 85%, more preferably more than 90%, and even more preferably more than 95%, 97% or even 99% identical to human ghrelin can be used for vaccination.

Guidance on how to modify human ghrelin or ghrelin peptide, in particular for binding to a virus-like particle, is given in this application. Immunization against ghrelin using compositions of the invention preferably containing human ghrelin or ghrelin peptide coupled to a core particle and VLP, respectively, may provide a method for treating obesity.

In a further highly preferred embodiment of the present invention, the antigen or antigenic determinant is ghrelin or ghrelin peptide comprising, or, alternatively, essentially consisting of, or, alternatively, consisting of an amino acid sequence selected from the group consisting of:

(a) GSSFLSPEHQRVQRKESKKPPAKLQPR (person) (SEQ ID No. 48)

(b) GSSFLSPEHQRVQQRKESKKPPAKLQPR (person) (SEQ ID No. 31)

(c) GSSFLSPEHQRVQ (person) (SEQ ID No. 60)

(d) QRKESKKPPAKLQPR (person) (SEQ ID No. 61)

(f) PPAKLQPR (person) (SEQ ID No. 53)

(f) GSSFLSPEHQKLQQRKESKKPPAKLQPR (dog) (SEQ ID No. 49)

(g) GSSFLSPEHQKLQRKESKKPPAKLQPR (dog) (SEQ ID No. 50)

(h) GSSFLSPEHQKLQ (dog) (SEQ ID No. 62)

(i) QRKESKKPPAKLQPR (dog) (SEQ ID No. 63)

(k) EHQRVQQRKE (person) (SEQ ID No. 56)

(l) GSSFLSPEHQKAQQRKESKKPPAKLQPR (mouse) (SEQ ID No. 32)

(n) the amino acid sequence of any fragment of any of (a) - (l)

In yet a further preferred embodiment of the present invention, the antigen or antigenic determinant further comprises at least one second attachment site selected from: (i) an attachment site not found in nature with said antigen or antigenic determinant, and (ii) an attachment site found in nature with the specified antigen or antigenic determinant. In a preferred embodiment, said attachment site comprises an amino acid linker of the invention, preferably a linker sequence of C, CG or CGG. Preferably, an antigen or antigenic determinant with said at least one second attachment site comprises, or, alternatively, essentially consists of, or, alternatively, consists of an amino acid sequence selected from the group consisting of:

(a) CGSSFLSPEHQRVQRKESKKPPAKLQPR (person) (SEQ ID No. 64)

(b) CGSSFLSPEHQRVQQRKESKKPPAKLQPR (person) (SEQ ID No. 65)

(c) GSSFLSPEHQRVQRKESKKPPAKLQPRC (person) (SEQ ID No. 66)

(d) GSSFLSPEHQRVQRKESKKPPAKLQPRGC (person) (SEQ ID No. 120)

(e) GSSFLSPEHQRVQQRKESKKPPAKLQPRC (person) (SEQ ID No. 67)

(f) GSSFLSPEHQRVQQRKESKKPPAKLQPRGC (person) (SEQ ID No. 121)

(g) GSSFLSPEHQRVQC (person) (SEQ ID No. 68)

(h) GSSFLSPEHQRVQGC (person) (SEQ ID No. 122)

(i) CQRKESKKPPAKLQPR (person) (SEQ ID No. 69)

(j) CPPAKLQPR (person) (SEQ ID No. 70)

(k) CGSSFLSPEHQKLQQRKESKKPPAKLQPR (dog) (SEQ ID No. 71)

(l) CGSSFLSPEHQKLQRKESKKPPAKLQPR (dog) (SEQ ID No. 72)

(m) GSSFLSPEHQKLQQRKESKKPPAKLQPRC (dog) (SEQ ID No. 73)

(n) GSSFLSPEHQKLQQRKESKKPPAKLQPRGC (dog) (SEQ ID No. 123)

(o) GSSFLSPEHQKLQRKESKKPPAKLQPRC (dog) (SEQ ID No. 74)

(p) GSSFLSPEHQKLQRKESKKPPAKLQPRGC (dog) (SEQ ID No. 124)

(q) GSSFLSPEHQKLQC (SEQ ID No. 75)

(r) GSSFLSPEHQKLQGC (SEQ ID No. 125)

(s) CEHQRVQQRKE (SEQ ID No. 76)

(t) CGSSFLSPEHQKAQQRKESKKPPAKLQPR (mouse) (SEQ ID No. 77)

(u) GSSFLSPEHQKAQRKESKKPPAKLQPRC (mouse) (SEQ ID No. 126)

(v) GSSFLSPEHQKAQRKESKKPPAKLQPRGC (mouse) (SEQ ID No. 127)

(w) GSSFLSPEHQKAQQRKESKKPPAKLQPRC (mouse) (SEQ ID No. 128)

(x) GSSFLSPEHQKAQQRKESKKPPAKLQPRGC (mouse) (SEQ ID No. 129)

(y) GSSFLSPEHQKAQC (mouse) (SEQ ID No. 130)

(z) GSSFLSPEHQKAQGC (mouse) (SEQ ID No. 131)

(aa) GGSSFLSPEHQGC (SEQ ID No. 132)

(bb) CKKPPAKLQPR (SEQ ID No. 133)

(cc) CEHQKAQQRKE (SEQ ID No. 134)

(dd) CEHQKAQQRKES (SEQ ID No. 135)

(ee) CLSPEHQKAQQ (SEQ ID No. 136)

(ff) CEHQRVQQRKES (SEQ ID No. 137)

(gg) CLSPEHQRVQQ (SEQ ID No. 138)

amino acid sequence of any fragment of any of (a) - (gg)

Some of the very preferred ghrelin peptides are described in Example 13, where, in particular, the corresponding murine peptides are described. These peptides as the second attachment site for binding to VLP and saws contain an N- or C-terminal cysteine residue. These very preferred short peptide fragments of ghrelin are capable of very high immunogenicity upon binding to VLP and core particles, respectively. In addition, the preferred fragments of ghrelin are also able to overcome the safety problems that arise when the target native proteins as a shorter fragment are much less likely to contain T-cell epitopes. As a rule, the shorter the peptides - the safer with respect to T-cell activation. However, peptides that are too short may not induce the induction of high affinity antibodies capable of firmly binding ghrelin in solution.

First, very preferred fragments of ghrelin peptides corresponding to the mouse ghrelin fragment from residues 1-10 (GSSFLSPEHQ) (SEQ ID No. 55) were selected, since the N-terminal ghrelin segment is identical in all known species. In addition, it is likely that it is identical in species where ghrelin is not yet identified. In addition, the C-terminal residue, glutamine, can increase solubility, facilitating the preparation of a soluble vaccine product upon binding to VLP and core particles, respectively. In fact, the solubility of the peptides is often a limiting factor for the binding efficiency and stability of the vaccine. Further considerations include the prevention of a potential T-cell epitope. Selecting a smaller fragment of the peptide reduces the likelihood of the presence of a T-cell epitope. The binding of residues 1-10 of mouse ghrelin through the C-terminus to VLP, upon immunization of mice, induces N-terminus-specific antibodies that can bind active ghrelin and, therefore, prevent its passage through the blood-brain barrier, leading to reduced food intake.

Additional very preferred fragments of ghrelin peptides corresponding to a fragment of a mouse ghrelin peptide from residues 42-51 (KKPPAKLQPR) (SEQ ID No. 52) were selected based on bases similar to those indicated above. The only difference is that this fragment of the peptide, connected through the N-terminus, is capable of inducing antibodies specific to the C-terminal part of ghrelin during immunization in mice. In addition, antibody recognition can be enhanced, since the n-octanoyl group on serine 3 can be avoided, hence reducing its ability to interfere. The binding of residues of 42-51 mouse ghrelin through the N-terminus to VLP induces antibodies that can neutralize active ghrelin and, therefore, prevent its passage through the blood-brain barrier, leading to reduced food intake.

In addition, several additional very preferred fragments of ghrelin peptides were selected corresponding to fragments of the mouse ghrelin peptide from residues 31-41 (EHQKAQQRKES) (SEQ ID No. 113) and residues 28-37 (LSPEHQKAQQ) (SEQ ID No. 117), covering the central segment ghrelin. Similarly, these fragments of ghrelin are devoid of potential T-cell epitopes, and also lack an n-octanoyl group on serine 3. The binding of these peptides through the N- or C-terminus to the VLP induces antibodies that can neutralize active ghrelin and, therefore, prevent its passage through blood-brain barrier, leading to reduced food intake.

Additional preferred ghrelin peptides suitable for use in the present invention can be identified by the use of existing or future monoclonal or polyclonal antibodies.

Additional preferred ghrelin molecules and peptides derived from molecules can be detected in nature in species where sequence information is not yet available.

One skilled in the art will understand that without deviating from the scope of the invention or any implementation thereof, it is easy to see and make other suitable modifications and adaptations to the methods and applications described herein. Now, after a detailed description of the present invention, the same will be more easily understood when referring to the following examples, included here only for the purpose of illustration and not intended to be a limitation of the invention.

EXAMPLES

EXAMPLE 1

Construction and expression of mutant Qβ coat proteins and purification of VLP or capsids from mutant Qβ coat proteins

Plasmid construction and cloning of mutant envelope proteins.

Construction of pQβ-240:

As the initial plasmid for constructing pQβ-240, the plasmid pQβ10 was used (Kozlovska, TM, et al., Gene 137: 133-137). Through reverse PCR, the mutation Lys13 → Arg was created. Inverted primers designed tail-to-tail in reverse directions:

5'-GGTAACATCGG TCG AGATGGAAAACAAACTCTGGTCC-3 '(SEQ ID No. 78)

and

5'-GGACCAGAGTTTTGTTTTCCATCT CGA CCGATGTTACC-3 '(SEQ ID No. 79).

The products of the first PCR were used as templates for the second PCR reaction in which a direct primer was used

5'-AGCTCGCCCGGGGATCCTCTAG-3 '(SEQ ID No. 80)

and reverse primer

5'-CGATGCATTTCATCCTTAGTTATCAATACGC-TGGGTTCAG3 '(SEQ ID No. 81).

The second PCR product was digested with XbaI and Mph1103I and cloned into the pQβ10 expression vector digested with the same restriction enzymes. PCR reactions were carried out with a set of reagents for PCR and according to the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using a direct label incorporation method confirmed the presence of the desired mutations. E. coli cells carrying pQβ-240 supported efficient synthesis of a 14 kDa protein co-migrating to SDS-PAGE with a control Qβ coat protein isolated from Qβ phage particles.

The resulting amino acid sequence: (SEQ ID No. 17)

AKLETVTLGNIG R DGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-243:

As the starting plasmid for constructing pQβ-243, the plasmid pQβ10 was used. By reverse PCR, the Asn10 → Lys mutation was created. Inverted primers designed tail-to-tail in reverse directions:

5'-GGCAAAATTAGAGACTGTTACTTTAGGTAA G ATCGG-3 '(SEQ ID No. 82)

and

5'-CCGAT C TTACCTAAAGTAACAGTCTCTAATTTTGCC-3 '(SEQ ID No. 83).

The products of the first PCR were used as templates for the second PCR reaction in which a direct primer was used

5'-AGCTCGCCCGGGGATCCTCTAG-3 '(SEQ ID No. 80)

and reverse primer

5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3 '(SEQ ID No. 81).

The second PCR product was digested with XbaI and Mph1103I and cloned into the pQβ10 expression vector digested with the same restriction enzymes. PCR reactions were carried out with a set of reagents for PCR and according to the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using a direct label incorporation method confirmed the presence of the desired mutations. E. coli cells carrying pQβ-243 supported efficient synthesis of a 14 kDa protein co-migrating to SDS-PAGE with a control Qβ coat protein isolated from phage Qβ particles.

The resulting amino acid sequence: (SEQ ID No. 18)

AKLETVTLG K IGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-250:

As the starting plasmid for constructing pQβ-250, the plasmid pQβ-240 was used. Through site-specific mutagenesis, a Lys2 → Arg mutation was created. For the synthesis of a mutant PCR fragment inserted at unique NcoI and HindIII restriction sites into the pQβ-185 expression vector, a direct primer was used

5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3 '(SEQ ID No. 84)

and reverse primer

5'-GATTTAGGTGACACTATAG-3 '(SEQ ID No. 85).

PCR reactions were carried out with a set of reagents for PCR and according to the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using a direct label incorporation method confirmed the presence of the desired mutations. E. coli cells carrying pQβ-250 supported efficient synthesis of a 14 kDa protein co-migrating to SDS-PAGE with a control Qβ envelope protein isolated from phage Qβ particles.

The resulting amino acid sequence: (SEQ ID No. 19)

A R LETVTLGNIG R DGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

Construction of pQβ-251:

As the starting plasmid for constructing pQβ-251, the plasmid pQβ10 was used. Through reverse PCR, the mutation Lys16 → Arg was created. Inverted primers were designed in reverse tail-to-tail directions:

5'-GATGGA CGT CAAACTCTGGTCCTCAATCCGCGTGGGG-3 '(SEQ ID No. 86)

and

5 ' -ACNACGCGGATTGAGGACCAGAGTTTG ACG TCCATC-3 '(SEQ ID No. 87).

The products of the first PCR were used as templates for the second PCR reaction in which a direct primer was used

5'-AGCTCGCCCGGGGATCCTCTAG-3 '(SEQ ID No. 80)

and reverse primer

5'-CGATGCATTTCATCCTTAGTTATCAATACGCTGGGTTCAG-3 '(SEQ ID No. 81).

The second PCR product was digested with XbaI and Mph1103I and cloned into the pQβ10 expression vector digested with the same restriction enzymes. PCR reactions were carried out with a set of reagents for PCR and according to the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using a direct label incorporation method confirmed the presence of the desired mutations. E. coli cells bearing pQβ-251 supported efficient synthesis of a 14 kDa protein co-migrating to SDS-PAGE with a control Qβ coat protein isolated from Qβ phage particles.

The resulting amino acid sequence encoded by this construct is shown in SEQ ID No. 20.

Construction of pQβ-259:

As the starting plasmid for constructing pQβ-259, the plasmid pQβ-251 was used. Through site-specific mutagenesis, a Lys2 → Arg mutation was created. For the synthesis of a mutant PCR fragment inserted at unique NcoI and HindIII restriction sites into the pQβ-185 expression vector, a direct primer was used

5'-GGCCATGGCACGACTCGAGACTGTTACTTTAGG-3 '(SEQ ID No. 84)

and reverse primer

5'-GATTTAGGTGACACTATAG-3 '(SEQ ID No. 85).

PCR reactions were carried out with a set of reagents for PCR and according to the manufacturer's protocol (MBI Fermentas, Vilnius, Lithuania).

Sequencing using a direct label incorporation method confirmed the presence of the desired mutations. E. coli cells carrying pQβ-259 supported efficient synthesis of a 14 kDa protein co-migrating to SDS-PAGE with a control Qβ coat protein isolated from phage Qβ particles.

The resulting amino acid sequence: (SEQ ID No. 21)

AKLETVTLGNIGKDGKQTLVLNPRGVNPTNGVASLSQAGAVP

ALEKRVTVSVSQPSRNRKNYKVQVKIQNPTACTANGSCDPSVTRQ

KYADVTFSFTQYSTDEERAFVRTELAALLASPLLIDAIDQLNPAY

The main methods of expression and purification of Qβ and Qβ mutants.

Cleaning

E. coli JM109 was transformed with a Qβ plasmid expressing a coat protein. 5 ml of LB liquid medium containing 20 μg / ml ampicillin was inoculated with clones transformed with plasmids expressing Qβ coat protein. The inoculated culture was incubated for 16-24 hours at 37 ° C without stirring. The resulting inoculum was subsequently diluted 1: 100 in 100-300 ml of fresh LB medium containing 20 μg / ml ampicillin and incubated overnight at 37 ° C without stirring. The resulting second inoculum was diluted 1:50 in M9 medium containing 1% casamino acids and 0.2% glucose in flasks and incubated overnight at 37 ° C with stirring.

Cleaning

Solutions and buffers for cleaning procedures

1. LB Lysis Buffer

50 mM Tris-HCl pH 8.0 with 5 mM EDTA, 0.1% TritonX100 and freshly prepared PMSF at a concentration of 5 micrograms per ml. Without lysozyme and DNase.

2. SAS

Saturated ammonium sulfate in water

3. Buffer NET.

20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and

150 mM NaCl.

4. PEG

40% (w / v) polyethylene glycol 6000 in NET

Destruction and lysis

Frozen cells were resuspended in LB at 2 ml / g cells. The mixture was sonicated five times for 15 seconds at 22 kHz at 1 minute intervals to cool the solution on ice. The lysate was then centrifuged for 1 hour at 14,000 rpm using a Janecki K 60 rotor. All centrifugation steps described below were carried out using the same rotor, unless otherwise indicated. The supernatant was stored at 4 ° C, while cell debris was washed twice with LB. After centrifugation, the lysate supernatants and the washed fractions were combined.

Fractionation

A saturated solution of ammonium sulfate was added dropwise to the lysate combined above with stirring. The SAS volume was adjusted to one fifth of the total volume to obtain 20% saturation. The solution was left overnight, and the next day was centrifuged for 20 minutes at 14,000 rpm. The precipitate was washed with a small amount of 20% ammonium sulfate and centrifuged again. The resulting supernatants were combined and SAS was added dropwise to obtain 40% saturation. The solution was left overnight, and the next day was centrifuged for 20 minutes at 14,000 rpm. The resulting precipitate was dissolved in NET buffer.

Chromatography

The capsid protein or VLP redissolved in NET buffer was placed on a CL-4B Sepharose column. Three peaks were eluted during chromatography. The first peak mainly contained membranes and membrane fragments, and it was not collected. Capsids were contained in the second peak, while the third peak contained other E. coli proteins.

Peak fractions were combined and the NaCl concentration was adjusted to a final concentration of 0.65 M. With stirring, a volume of PEG solution corresponding to half of the combined peak fraction was added dropwise. The solution was left overnight without stirring. Capsid protein was precipitated by centrifugation for 20 minutes at 14,000 rpm. Then it was dissolved in a minimal volume of NET and again placed on a CL-4B Sepharose column. Peak fractions were combined and precipitated with ammonium sulfate at 60% saturation (w / v). After centrifugation and redissolution in NET buffer, the capsid protein was placed on a CL-4B Sepharose column for rechromatography.

Dialysis and drying

The peak fractions obtained above were combined and subjected to intensive dialysis against sterile water and lyophilized for storage.

Expression and purification of Qβ-240

Cells ( E. coli JM 109 transformed with pQβ-240 plasmid) were resuspended in LB, sonicated five times for 15 seconds (in an ice-water bath), and centrifuged for 13 hours at 13,000 rpm. Supernatants were stored at 4 ° C until further processing, while debris was washed 2 times with 9 ml of LB, and finally 9 ml of 0.7 M urea in LB. All supernatants were combined and placed on a CL-4B Sepharose column. The collected peak fractions were precipitated with ammonium sulfate and centrifuged. Then the redissolved protein was further purified on a column with Sepharose 2B, and finally on a column with Sepharose 6B. The capsid peak was finally subjected to intensive dialysis against water and lyophilized as described above. The assembly of the shell protein into the capsid was confirmed by electron microscopy.

Expression and purification of Qβ-243

Cells ( E. coli RR1) were resuspended in LB and processed as described in the main procedure. The protein was purified by two successive gel filtration steps on a CL-4B Sepharose column and finally on a CL-2B Sepharose column. Peak fractions were combined and lyophilized as described above. The assembly of the shell protein into the capsid was confirmed by electron microscopy.

Expression and purification of Qβ-250

Cells ( E. coli JM109 transformed with Qβ-250) were resuspended in LB and processed as described in the main procedure. The protein was purified by gel filtration on a CL-4B Sepharose column and finally on a CL-2B Sepharose column and lyophilized as described above. The assembly of the shell protein into the capsid was confirmed by electron microscopy.

Expression and purification of Qβ-259

Cells ( E. coli JM109 transformed with Qβ-259) were resuspended in LB and sonicated. Debris was washed once with 10 ml of LB and a second time with 10 ml of 0.7 M urea in LB. The protein was purified by two successive gel filtration steps on a CL-4B Sepharose column. The protein was dialyzed and lyophilized as described above. The assembly of the shell protein into the capsid was confirmed by electron microscopy.

EXAMPLE 2

The insert containing a lysine residue of the peptide in the epitope c / e1 HBcAg (1-149)

Epitope c / e1 (residues 72 to 88) of HBcAg is located in the upper region on the surface of the hepatitis B virus capsid (HBcAg). Part of this region (proline 79 and alanine 80) was genetically replaced by the Gly-Gly-Lys-Gly-Gly peptide (SEQ ID No. 33), which led to the construction of HBcAg-Lys (SEQ ID No. 26). The introduced lysine residue contains a reactive amino group on its side chain, which can be used for intramolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group.

HBcAg-Lys DNA with the amino acid sequence shown in SEQ ID No. 78 was obtained by PCR: Two fragments encoding HBcAg fragments (amino acid residues 1 to 78 and 81 to 149) were separately amplified by PCR. The primers used for these PCRs also introduced the DNA sequence encoding the Gly-Gly-Lys-Gly-Gly peptide (SEQ ID No. 33). The HBcAg fragment (1 to 78) was amplified with pEco63 using EcoRIHBcAg (s) and Lys-HBcAg (as) primers. The HBcAg fragment (81 to 149) was amplified with pEco63 using Lys-HBcAg (s) and HBcAg (1-149) Hind (as) primers. The primers Lys-HBcAg (as) and Lys-HBcAg (s) at the ends of the PCR products were introduced into complementary DNA sequences, allowing the fusion of these two PCR products during subsequent combining PCR. The collected fragments were amplified by PCR using EcoRIHBcAg (s) and HbcAg (1-149) Hind (as) primers.

For PCR, 100 pmol of each oligonucleotide and 50 ng of template DNA were used in 50 ml of reaction mixtures with 2 units of Pwo polymerase, 0.1 mM dNTP and 2 mM MgSO ₄ . For both reactions, a cyclic change in temperature was carried out as follows: 2 minutes - 94 ° C; 30 cycles consisting of 94 ° C (1 minute), 50 ° C (1 minute), 72 ° C (2 minutes).

Primer Sequence:

EcoRIHBcAg (s):

(5'-CCGGAATTCATGGACATTGACCCTTATAAAG-3 ') (SEQ ID No. 88);

Lys-HBcAg (as):

(5'-CCTAGAGCCACCTTTGCCACCATCTTCTAAATTAGTACCCACCCAGAG

GTAGC-3 ') (SEQ ID No. 89);

Lys-HBcAg (s):

(5'-

GAAGATGGTGGCAAAGGTGGCTCTAGGGACCTAGTAGTCAGTTAT

GTC-3 ') (SEQ ID No. 90);

HBcAg (1-149) Hind (as):

(5'-CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAG-3 ') (SEQ ID No. 91).

To fuse the two PCR fragments, 100 pmol of EcoRIHBcAg (s) and HBcAg (1-149) Hind (as) primers with 100 ng of two purified PCR fragments in 50 ml of the reaction mixture containing 2 Pwo polymerase units, 0.1 mM dNTP and 2 mM MgSO ₄ . The parameters of the PCR cycles were: 94 ° C - 2 minutes; 30 cycles consisting of 94 ° C (1 minute), 50 ° C (1 minute), 72 ° C (2 minutes). The combined PCR product was analyzed by agarose gel electrophoresis, purified and digested with restriction enzymes EcoRI and HindIII for 19 hours in an appropriate buffer. The digested DNA fragments were ligated into the digested EcoRI / HindIII pKK vector to obtain the pKK-HBcAg-Lys expression vector. The insertion of the PCR product into the vector was analyzed by restriction analysis of EcoRI / HindIII and the determination of the sequence of DNA insertion.

EXAMPLE 3

Expression and purification of HBcAg-Lys.

The E. coli strains K802 or JM109 were transformed with pKK-HBcAg-Lys. 1 ml of overnight bacterial culture was used to inoculate 100 ml of LB medium containing 100 μg / ml ampicillin. This culture was grown for 4 hours at 37 ° C until the OD at 600 nm reached approximately 0.8. Induction of HBcAg-Lys synthesis was performed by adding IPTG to a final concentration of 1 mM. After induction, the bacteria were further stirred for 4 hours at 37 ° C. Bacteria were collected by centrifugation for 15 minutes at 5000 × g. The precipitate was frozen at -80 ° C. The pellet was thawed and resuspended in bacterial lysis buffer (10 mM Na ₂ HPO ₄ , pH 7.0, 30 mM NaCl, 0.25% Tween-20, 10 mM EDTA), supplemented with 200 μg / ml lysozyme and 10 μl benzoase ( Merck). Cells were incubated for 30 minutes at room temperature and destroyed by sonication. To induce the expression of HBcAg-Lys via IPTG, E. coli cells carrying the pKK-HBcAg-Lys expression plasmid or a control plasmid were used. Before IPTG was added, samples were taken from a bacterial culture bearing the pKK-HBcAg-Lys plasmid and from a culture carrying the control plasmid. Four hours after the addition of IPTG, samples were again taken from the bacterial culture containing the plasmid pKK-HBcAg-Lys. Protein expression was monitored by SDS-PAGE followed by Coomassie staining.

Then the lysate was centrifuged for 30 minutes at 12,000 × g to remove insoluble cell debris. The supernatant and pellet were analyzed by Western blotting using a monoclonal antibody against HBcAg (YVS1841, obtained from Accurate Chemical and Scientific Corp., Westbury, NY, USA), indicating that a significant amount of HBcAg-Lys protein was soluble. Briefly, lysates from E. coli cells expressing HBcAg-Lys and from control cells were centrifuged at 14,000 × g for 30 minutes. The supernatant (= soluble fraction) and the precipitate (= insoluble fraction) were separated and diluted with sample SDS buffer to equal volumes. Samples were analyzed by SDS-PAGE followed by western blotting with monoclonal antibody against HBcAg YVS 1841.

The purified cell lysate was used for centrifugation in a stepwise gradient using a stepwise sucrose gradient consisting of 4 ml of a 65% sucrose solution, with layered 3 ml of a 15% sucrose solution, followed by 4 ml of bacterial lysate. The sample was centrifuged for 3 hours at 100,000 × g at 4 ° C. After centrifugation, 1 ml of fractions was collected on top of the gradient and analyzed by SDS-PAGE followed by Coomassie staining. The HBcAg-Lys protein was detected by Coomassie staining.

Protein HBcAg-Lys was enriched at the interface of 15 and 65% sucrose, meaning that it formed a capsid particle. Most bacterial proteins remained in the upper layer of the gradient without sucrose; therefore, centrifugation of HBcAg-Lys particles in a stepwise gradient led to enrichment and partial purification of the particles.

Expression and purification of HBcAg-Lys were generally performed as follows. An overnight culture was prepared by inoculating one colony in 100 ml LB, 100 μg / ml ampicillin and growing the culture overnight at 37 ° C. 25 ml of the preliminary culture the next day was diluted with 800 ml of LB medium with ampicillin and the culture was grown to an optical density of OD ₆₀₀ equal to 0.6-0.8. The culture was then induced with 1 mM IPTG and allowed to grow for another 4 hours. Cells were harvested and lysed essentially as described above.

Then, HBcAg-Lys was purified by first precipitating the protein from the purified cell lysate in ammonium sulfate (30% saturation), then loading the re-dissolved precipitate onto a gel filtration column (Sephacryl S-400, Pharmacia). The combined fractions were again precipitated with ammonium sulfate, re-dissolved and placed on the same gel filtration column a second time. Finally, the fractions were collected and concentrated, and the concentrations were evaluated using the Bradford method (BioRad).

EXAMPLE 4

Design devoid of cysteine residues and containing inserted lysine residue HBcAg

Hepatitis Cortical Antigen (HBcAg), referred to herein as HBcAg-lys-2cys-Mut, lacking cysteine residues at positions corresponding to 48 and 107 in SEQ ID No. 25 and containing an inserted lysine residue, was constructed using the following methods.

Through the first separate amplification of three fragments of the HBcAg-Lys gene, obtained as described above in example 2, with the following combinations of PCR primers, two mutations were introduced. To obtain the HBcAg-lys-2cys-Mut gene, PCR methods and conventional cloning methods were used.

Briefly, to obtain fragment 1, the following primers were used:

Primer 1: EcoRIHBcAg (s)

CCGGAATTCATGGACATTGACCCTTATAAAG (SEQ ID No. 88)

Primer 2: 48as

GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC (SEQ ID No. 92)

To obtain fragment 2, the following primers were used:

Primer 3: 48s

GAGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC (SEQ ID No. 93)

Primer 4: 107as

CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC (SEQ ID No. 94)

To obtain fragment 3, the following primers were used:

Primer 5: HBcAg149hind-as

CGCGTCCCAAGCTTCTAAACAACAGTAGTCTCCGGAAGCGTTGATAG (SEQ ID No. 95)

Primer 6: 107s

GTGGTTTCACATTTCTTCCCTCACTTTTGGAAG (SEQ ID No. 96)

Fragments 1 and 2 were then combined with EcoRIHBcAg (s) and 107as PCR primers to obtain fragment 4. Then, fragment 4 and fragment 3 were combined with EcoRIHBcAg (s) and HBcAg149hind-as primers to obtain the full-sized gene. The full-sized gene was then digested with EcoRI (GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK vector (Pharmacia) digested at the same restriction sites. Expression and purification of HBcAg-lys-2cys-Mut was performed as described in example 3.

EXAMPLE 5

Construction of HBcAg1-185-Lys.

Hepatitis Cortical Antigen (HBcAg) 1-185 was modified as described in Example 2. A part (proline 79 and alanine 80) of the c / e1 epitope region (residues 72 to 88) was genetically replaced by the Gly-Gly-Lys-Gly-Gly peptide ( SEQ ID No. 33), resulting in the construction of HBcAg-Lys (SEQ ID No. 26). The introduced lysine residue contains a reactive amino group on its side chain, which can be used for intramolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group. To obtain the HBcAg1-185-Lys gene, PCR methods and traditional cloning methods were used.

The Gly-Gly-Lys-Gly-Gly sequence (SEQ ID No. 33) was inserted by amplification of two separate fragments of the HBcAg gene with pEco63, as described above in Example 2, and subsequent fusion of the two fragments by PCR to assemble the full-sized gene. Used the following combinations of primers for PCR:

Fragment 1:

Primer 1: EcoRIHBcAg (s) ((SEQ ID No. 88), see Example 2)

Primer 2: Lys-HBcAg (as) ((SEQ ID No. 89), see example 2)

Fragment 2:

Primer 3: Lys-HBcAg (s) ((SEQ ID No. 90), see example 2)

Primer 4: HBcAgwtHindIIII

CGCGTCCCAAGCTTCTAACATTGAGATTCCCGAGATTG (SEQ ID No. 97)

Assembly:

Primer 1: EcoRIHBcAg (s) ((SEQ ID No. 88), see Example 2)

Primer 2: HBcAgwtHindIIII (SEQ ID No. 97)

The assembled full-size gene was then digested with the EcoRI (GAATTC) and HindIII (AAGCTT) enzymes and cloned into the pKK (Pharmacia) vector digested at the same restriction sites.

EXAMPLE 6

Fusion of the peptide epitope in the MIR region of HBcAg.

Residues HBcAg1-185 79 and 80 were replaced by the CεH3 epitope with the sequence VNLTWSRASG (SEQ ID No. 98). The CεH3 sequence is derived from the sequence of the third constant domain of the heavy chain of human IgE. The epitope was inserted into the HBcAg1-185 sequence using a PCR combining method. In the first PCR step, the HBcAg1-185 gene, derived from the ATCC pEco63 clone and amplified with HBcAg-wt EcoRI fwd and HBcAg-wt Hind III rev primers, was used as a template in two separate reactions to amplify two fragments containing elements of a sequence encoding a sequence CεH3. Then these two fragments were combined in the second stage of PCR, in a unifying PCR reaction.

Combinations of primers in the first stage of PCR: СεР3fwd with HBcAg-wt HindIII rev and HBcAg-wt EcoRI fwd with СεР3rev. In the PCR combining reaction, two fragments isolated in the first PCR step were first combined for 3 PCR cycles without external primers, which were subsequently added to the reaction mixture for the next 25 cycles. External primers: HBcAg-wt EcoRI fwd and HBcAg-wt Hind III rev.

The PCR product was cloned into pKK223.3 using EcoRI and HindIII sites for expression in E. coli (see Example 2). Hybrid VLPs were expressed in E. coli and purified as described in Example 2. The elution volume at which HBcAg1-185-CεP3 was eluted by gel filtration showed the assembly of the fusion proteins into the hybrid VLP.

Primer Sequences:

CεH3fwd:

5'GTT AAC TTG ACC TGG TCT CGT GCT TCT GGT GCA TCC AGG GAT CTA GTA GTC 3 '(SEQ ID No. 99)

VNLTWSRASG A80 SRDLV V86 (SEQ ID No. 100)

CεH3rev:

5 'ACC AGA AGC ACG AGA CCA GGT CAA GTT AAC ATC TTC CAA ATT ATT ACC CAC 3' (SEQ ID No. 101)

D78 E L N N G V72 (SEQ ID No. 102)

HBcAg-wt EcoRI fwd:

5 ' CCG gaattcATGGACATTGACCCTTATAAAG (SEQ ID No. 103)

HBcAG-wt Hind III rev:

5 ' CGCGTCCC aagcttCTAACATTGAGATTCCCGAGATTG (SEQ ID No. 104)

EXAMPLE 7

The fusion of the epitope of the ghrelin peptide with the MIR region of HBcAg.

Residues 79 and 80 of HBcAg1-185 were replaced by an epitope of a ghrelin peptide with the sequence: EHQRVQQRKE (SEQ ID No. 56). Two overlapping primers were constructed using the same strategy as described in Example 6, and a fusion protein was constructed by combining PCR. The PCR product was cloned into pKK223.3 vector and expressed in E. coli K802. Hybrid VLPs were expressed and purified as described in Example 3.

EXAMPLE 8

The fusion of the epitope of the ghrelin peptide with the C-terminus of the shortened at 19 position CP extension of A1 Qβ protein.

In the PCR reaction with pQβ10, a primer annealed to the 5'-end of the A1 Qβ gene and a primer annealed to the 3'-end of the A1 Qβ gene and further containing a sequence element encoding a ghrelin fragment with the KKPPAKLQPR sequence (SEQ ID No. 52) were used as a template. ) The PCR product was cloned into pQβ10 (Kozlovska T. M. et al., Gene 137: 133-37 (1993)), and the hybrid VLP was expressed and purified as described in Example 1.

EXAMPLE 9

Insertion of the epitope of the ghrelin peptide between positions 2 and 3 of the coat protein fr.

In the Bsp119I region of the vector pFrd8 (Pushko, P. et al., Prot. Eng. 6: 883-91 (1993)), complementary primers encoding the ghrelin peptide sequence with the sequence EHQRVQQRKE (SEQ ID No. 56) and were inserted using standard molecular biology methods containing Bsp119I- compatible ends and additional nucleotides nucleotides, enabling insertion while maintaining the reading frame. Alternatively, the “sticky ends” of the pFrd8 vector after Bsp119I cleavage were supplemented with a Klenov fragment and oligonucleotides encoding the mouse ghrelin peptide sequence of the mouse ghrelin peptide and additional nucleotides for cloning with preservation of the reading frame after processing with the Klenov fragment were ligated into pFrd8. Clones with an insert in the right orientation were analyzed by sequencing. Expression and purification of the chimeric fusion protein in JM109 E. coli or K802 E. coli was performed as described in Pushko, P. et al, Prot. Eng. 6: 883-91 (1993), except for chromatographic steps that were performed using Sepharose CL-4B or Sephacryl S-400 (Pharmacia). The cell lysate was precipitated with ammonium sulfate and purified by two successive gel filtration purification steps, similar to the procedure described for Qβ in Example 1.

EXAMPLE 10

Insertion of the epitope of the ghrelin peptide between positions 67 and 68 of the p1 Ty1 protein in the pOGS8111 vector.

Synthesized two complementary nucleotides encoding the sequence EHQRVQQRKE (SEQ ID No. 56) of the peptide of human ghrelin, with ends compatible with the NheI site pOGS8111. Additional nucleotides were added to ensure insertion without shifting the reading frame of the sequence encoding the mouse ghrelin epitope as described in EP06777111. The amino acids AS and SS flanking the inserted epitope are encoded by altered NheI sites, which results from the insertion of the oligonucleotide into the TyA (d) pOGS8111 gene.

POGS8111 was transformed into S. cervisiae MC2 strain for the expression of a hybrid VLP Ty, as described in EP0677111 and indicated here. Hybrid VLP Ty was purified by sucrose gradient ultracentrifugation as described in EP0677111.

EXAMPLE 11

Insertion of an epitope of ghrelin peptide into the main papilloma virus type 1 capsid protein L1 (BPV-1).

The sequence encoding the ghrelin epitope with the sequence EHQRVQQRKE (SEQ ID No. 56) was replaced by the sequence encoding amino acids 130-136 of the BPV-1 L1 gene cloned into the pFastBac1 vector (GIBCO / BRL) as described (Chackerian, B. et al., Proc. Natl. Acad. USA 96: 2373-2378 (1999)). The sequence of the construct was verified by nucleotide sequence analysis. Using the GIBCO / BRL baculovirus system, a recombinant baculovirus was obtained as described by the manufacturer. Hybrid VLPs were purified from baculovirus-infected Sf9 cells as described by Kirnbauer, R. et al., Proc. Natl. Acad. Sci. 89: 12180-84 (1992) and Greenstone, H. L., et al., Proc. Natl. Acad. Sci. 95: 1800-05 (1998).

EXAMPLE 12

Immunization of mice fused with ghrelin VLP peptides.

Hybrid VLPs representing the mouse ghrelin epitope with the sequence EHQRVQQRKE (SEQ ID No. 56) obtained in Examples 7, 9, 10 and 11 were used to immunize mice as described in Example 15. The serum obtained from immunized mice was analyzed in a ghrelin-specific ELISA as described in example 14.

The effect of the vaccine was checked by subsequent weight gain in mice and by measuring food intake.

EXAMPLE 13

Binding of mouse ghrelin and mouse ghrelin peptides to Qβ capsid protein: vaccines of ghrelin peptides.

The following ghrelin peptides were chemically synthesized:

cGhrel: CGSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID No. 77)

GhreIC: GSSFLSPEHQKAQQRKESKKPPAKLQPRGC (SEQ ID No. 105)

cGhrQ14: CGSSFLSPEHQKAQRKESKKPPAKLQPR (SEQ ID No. 106)

GhrQ14C: GSSFLSPEHQKAQRKESKKPPAKLQPRGC (SEQ ID No. 107)

Ghrel24-33C GGSSFLSPEHQGC (SEQ ID No. 132)

cGhrel42-51 CKKPPAKLQPR (SEQ ID No. 133)

cGhrel31-40 CEHQKAQQRKE (SEQ ID No. 134)

cGhrel31-41 CEHQKAQQRKES (SEQ ID No. 135)

cGhrel28-37 CLSPEHQKAQQ (SEQ ID No. 136)

containing a cysteine residue added at the N- or C-terminus for binding to VLP or saws and used for chemical binding to Qβ, as described below.

A solution of 5 ml of 140 mM Qβ capsid protein in 20 mM Hepes, 150 mM NaCl pH 7.4 for 30 minutes was reacted with 108 μl of a 65 mM solution of SMPH (Pierce) in H ₂ O at 25 ° C on a shaking shaker. The reaction solution is then dialyzed twice for 2 hours against 5 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C. Then, 100 μl of the dialyzed reaction mixture was reacted with either 28.6 μl of a 10 mM stock solution (in DMSO) of ghrelin peptide (Qβ peptide / capsid protein ratio of 1:10). The binding reaction is carried out for 2 hours at 15 ° C in a water bath. Then, the reaction mixture was dialyzed for 24 hours against 2 × 5 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C.

The binding products are centrifuged, and supernatants and precipitates are analyzed on 16% SDS-PAGE gels under reducing conditions. The gels are stained with Coomassie brilliant blue.

Binding of ghrelin peptides to capsid protein fr

A solution of 120 mM fr capsid protein in 20 mM Hepes, 150 mM NaCl pH 7.2, was reacted with a 10-fold molar excess of SMPH (Pierce) diluted from the mother liquor in DMSO at 25 ° C. on a shaking shaker. The reaction solution is then dialyzed twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C. The dialyzed fr reaction mixture is then reacted overnight at 16 ° C on a shaker with an equimolar concentration of ghrelin peptide or with a ghrelin / fr peptide ratio of 1: 2. Binding products are analyzed by SDS-PAGE.

Binding of ghrelin peptides to HBcAg-Lys-2cys-Mut

A solution of 120 mM HBcAg-Lys-2cys-Mut in 20 mM Hepes, 150 mM NaCl, pH 7.2, is reacted with a 10-fold molar excess of SMPH (Pierce)) diluted from the mother liquor in DMSO at 25 ° C. swinging shaker. The reaction solution is then dialyzed twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C. Then the dialyzed HBcAg-Lys-2cys-Mut reaction mixture was reacted overnight at 16 ° C on a shaking shaker with an equimolar concentration of ghrelin peptide or with a ghrelin / HBcAg-Lys-2cys-Mut peptide ratio of 1: 2. Binding products are analyzed by SDS-PAGE.

Binding of ghrelin peptides to pyls

A solution of 125 mM E. coli type 1 pili in 20 mM Hepes, pH 7.2-60 minutes was reacted with a 50-fold molar excess of crosslinking agent SMPH (Pierce)) diluted from the mother liquor in DMSO, with RT on a shaking shaker. The reaction mixture was desalted on a PD-10 column (Amersham-Pharmacia Biotech). Protein-containing fractions were collected that eluted from the column, and the desalted derivatized pili protein overnight at 16 ° C on a shaking shaker was reacted with ghrelin peptides in an equimolar ratio or in a peptide to saw ratio of 1: 2. Binding products are analyzed by SDS-PAGE.

EXAMPLE 14

Immunization of mice with ghrelin VLP peptide conjugates.

Immunization of mice with Qβ-linked ghrelin peptides

Female C57BL / 6 mice are immunized with the mouse ghrelin peptides listed in Example 13, bound to Qβ, mixed with alum, dissolved in IFA, or without adjuvant. 50 μg are injected subcutaneously on days 0, 14 and 28. After immunization, blood was collected retro-orbitally in mice on days 14, 21, 28, 42, and then at monthly intervals thereafter. Serum was analyzed using peptide-specific ghrelin ELISA.

EXAMPLE 15

Binding of C-ghrelin and ghrelin-GC to the Qβ coat protein

2 ml of a solution of 2.0 mg / ml capsid protein Qβ in 20 mM Hepes, 150 mM NaCl pH 7.2 30 minutes at 25 ° C was reacted with 114.4 μl SMPH solution (Pierce) (from a 50 mM stock solution diluted DMSO). The reaction solution was then dialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C.

Then, the dialyzed derivatized Qβ VLP was reacted with mouse C-ghrelin peptide (SEQ ID: No. 77) or mouse ghrelin-GC (SEQ ID: No. 105) for 2 hours at 15 ° C in 20 mM Hepes, 150 mM NaCl, pH 7.2. The binding reaction was carried out in several aliquots, where 1 ml of derivatized VLP Qβ (at a concentration of 2 mg / ml) was reacted with 286 μl of a 10 mM peptide solution. 10% acetonitrile was added for the coupling reaction. The reaction solution was dialyzed once for 2 hours and then overnight against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 ° C.

Binding products were analyzed on 16% SDS-PAGE gels. The gels were stained with Coomassie brilliant blue. The results are shown in Figure 1. Binding products can be detected in Coomassie stained gels and clearly demonstrate covalent binding of C-ghrelin or ghrelin-GC to the capsid protein Qβ. Bands corresponding to the binding of one, two, three or four peptides associated with one subunit are indicated by arrows.

The figure 1 presents the binding products of the reaction of mouse C-ghrelin or mouse ghrelin-GC with the capsid protein Qβ. Lanes 2 and 4 show the binding products in the soluble fraction of Qβ-C-ghrelin and ghrelin-GC-Qβ, respectively. There is very little product in the insoluble fraction.

EXAMPLE 16

Immunization of mice with Qβ capsid protein bound C-ghrelin or Ghrelin-GC

Female C57BL / 6 mice (5 per group) were vaccinated with either mouse C-ghrelin (SEQ ID: No. 77) or mouse ghrelin-GC (SEQ ID: No. 105) associated with the Qβ capsid protein. 50 μg of the dialyzed vaccine from each sample was diluted in PBS to a volume of 200 μl and injected subcutaneously (100 μl from both sides of the abdomen) on days 0, 14 and 42. The vaccine was administered without additional adjuvant. As a control, a group of mice were immunized with PBS. Blood was collected retroorbitally in mice on days 0, 14, 21, 42, and their serum was analyzed using ghrelin-specific ELISA (described in Example 17). In addition, in mice during the experiment through regular controlled body weight and food intake (described in example 18).

EXAMPLE 17

Identification of ghrelin-specific antibodies in ELISA

ELISA plates were coated with mouse ghrelin with octanoylated serine (Bachem, Product No. H-4862) at a concentration of 20 μg / ml. The plates were blocked and then incubated with serially diluted murine sera from 14, 21, and 42 days. Antibody binding was detected using enzymatically labeled anti-mouse IgG antibodies. As a control, serum was also tested prior to immunization (Figure 2).

In mice immunized with Qβ-C-ghrelin or ghrelin-CG-Qβ, the average titer on day 21 reached 13,000 and 8,000, respectively (Figure 2). Serum prior to immunization or serum from mice immunized with PBS did not show any activity against ghrelin. This clearly demonstrates that the ghrelin-VLP conjugate is able to induce a high titer of antibodies against ghrelin, even if it is its own protein.

EXAMPLE 18

The effectiveness of experiments with C-ghrelin or ghrelin-GC associated with the capsid protein Qβ

Female C57BL / 6 mice (5 per group) were vaccinated with mouse C-ghrelin or ghrelin-GC coupled to the Qβ capsid protein as described in Example 16. As a control, a group of mice were immunized with PBS. All mice between days 0 and 14 were kept on a normal diet. Then, all mice were fed a high fat (45%) diet to facilitate the development of diet-induced obesity. Food and water were administered freely. In individual mice, changes in body weight were monitored, and food and water consumption per cell were monitored at regular intervals after immunization (on days 5, 11, 14, 21, 28, 35, 40, 49, and 55 after immunization) (i.e. group) (see figure 3).

In mice immunized with murine Qβ-C-ghrelin or mouse ghrelin-CG-Qβ, an average decrease in food intake of about 30-40% was achieved compared to control mice immunized with PBS (Figure 3). This clearly demonstrates that the ghrelin-VLP conjugate is able to suppress appetite and reduce food intake.

EXAMPLE 19

Efficacy of experiments with C-ghrelin or ghrelin-GC associated with Qβ capsid protein in an animal model of obesity

Female C57BL / 6 ob / ob mice (5 per group) were vaccinated with murine C-ghrelin or ghrelin-GC bound to the Qβ capsid protein. Control mice were immunized with PBS or QL VLP alone. 50 μg of the dialyzed vaccine or control protein is dissolved in 200 μl of PBS and injected subcutaneously (100 μl on both sides of the abdomen) with or without alum for 0 days. Mice were subjected to secondary stimulation with the appropriate drug on days 14, 28 and 42.

In mice, retroorbital blood is collected on days 0, 14, 21, 42, 56, 70, and then at monthly intervals. Sera are analyzed for the presence of ghrelin-specific antibodies in a ghrelin-specific ELISA, as described in Example 17. If the titers of ghrelin-specific antibodies are significantly reduced during the experiment period, the mice are respectively subjected to secondary stimulation. The effect of the vaccine was checked by measuring food intake in the absence of restrictions and the subsequent increase in the mass of mice, as described in example 18.

EXAMPLE 20

AP205 Shell Protein Gene Cloning

envelope protein cDNA (CP) AP205 (SEQ ID NO: 28) was collected from two cDNA fragments obtained from RNA of AP205 phage using reverse transcription-PCR and cloning into a commercial plasmid pCR 4-TOPO to determine the sequence. Reverse transcription methods are well known to those skilled in the art. The first fragment, contained in plasmid p205-246, contained 269 nucleotides upstream of the CP sequence and 74 nucleotides encoding the first 24 N-terminal amino acids of CP. The second fragment, contained in plasmid p205-262, contained 364 nucleotides encoding amino acids l2-131 CP and an additional 162 nucleotides downstream of the CP sequence. Both p205-246 and p205-262 were a generous gift from J. Klovins.

To fuse both CP fragments from plasmids p205-246 and p205-262 into one full-length CP sequence, plasmid 283.-58 was constructed using two-stage PCR.

The direct primer p1.44 containing the NcoI region for cloning into plasmid pQb185 or p1.45 containing the XbaI region for cloning into plasmid pQb10 and the reverse primer p1.46 containing the HindIII restriction region (sequences recognized by restriction enzymes are underlined) were tried :

p1.44 5'-AA CCATGG CAAATAAGCCAATGCAACCG-3 '(SEQ ID No. 139)

p1.45 5'-AA TCTAGA ATTTTCTGCGCACCCATCCCGG-3 '(SEQ ID No. 140)

p1.46 5'-AA AAGCTT AAGCAGTAGTATCAGACGATACG-3 '(SEQ ID No. 141)

Two additional primers were used to amplify fragments of the first PCR: p1.47, annealed to the 5'-end of the fragment contained in p205-262, and p1.48, annealed to the 3'end of the fragment contained in plasmid p205-246. Primers p1.47 and p1.48 are complementary to each other.

p1.47: 5'-GAGTGATCCAACTCGTTTATCAACTACATTTTCAGCAAGTCTG-3 '(SEQ ID No. 142)

p1.48: 5'-CAGACTTGCTGAAAATGTAGTTGATAAACGAGTTGGATCACTC-3 '(SEQ ID No. 143)

In the first two PCR reactions, two fragments were obtained. The first fragment was obtained with primers p1.45 and p1.48 and matrix p205-246. The second fragment was obtained with primers p1.47 and p1.46, and matrix p205-262. Both fragments were used as templates for the second PCR reaction, expansion of splicing-overlapping, with a combination of primers p1.45 and p1.46 or p1.44 and p1.46. The product of two second-stage PCR reactions was digested with XbaI or NcoI, respectively, and HindIII, and cloned into the same restriction sites in pQb10 or pQb185, respectively, two expression vectors derived from pGEM under the control of the E. coli tryptophan operon promoter.

Two plasmids were obtained, pAP283-58 (SEQ ID No. 27) containing the gene encoding CP wt AP205 (SEQ ID No. 28) in pQb10, and pAP281-32 (SEQ ID No. 30) with the mutation Pro5 → Thr (SEQ ID No. 29 ), in pQbl85. Sequence of envelope proteins was checked by determining the DNA sequence. PAP283-58 contained 49 nucleotides upstream of the ATG CP codon, downstream of the XbaI site and contained the putative initial attachment site of the membrane protein mRNA ribosome attachment.

EXAMPLE 21:

Expression and purification of recombinant VLP AP205

A. Expression of Recombinant VLP AP205

E. coli JM109 was transformed with the plasmid pAP283-58. 5 ml of LB liquid medium with 20 μg / ml ampicillin was inoculated with one colony and incubated for 16-24 hours at 37 ° C without stirring.

The resulting inoculum was diluted 1: 100 in 100-300 ml of LB medium containing 20 μg / ml ampicillin and incubated at 37 ° C overnight without stirring. The resulting second inoculum was diluted 1:50 in 2TY medium containing 0.2% glucose and phosphate for buffering and incubated overnight at 37 ° C on a shaker. Cells were harvested by centrifugation and frozen at -80 ° C.

B. Purification of Recombinant VLP AP205

Solutions and buffers:

Lysis buffer

50 mM Tris-HCl pH 8.0 with 5 mM EDTA, 0.1% TritonX100 and PMSF at 5 micrograms per ml.

SAS

Saturated ammonium sulfate in water

Buffer NET .

20 mM Tris-HCl, pH 7.8 with 5 mM EDTA and

150 mM NaCl.

Peg

40% (w / v) polyethylene glycol 6000 in NET

Lysis

Frozen cells were resuspended in lysis buffer at 2 ml / g cells. The mixture was sonicated five times for 15 seconds at 22 kHz at 1 minute intervals to cool the solution on ice. Then the lysate was centrifuged for 20 minutes at 12,000 rpm using the F34-6-38 rotor (Eppendorf). All centrifugation steps described below were carried out using the same rotor, unless otherwise indicated. The supernatant was stored at 4 ° C, while cell debris was washed twice with lysis buffer. After centrifugation, the lysate supernatants and the washed fractions were combined.

Ammonium sulfate precipitation can additionally be used to clean the VLP AP205. In a first step, a concentration of ammonium sulfate is selected at which VLP AP205 does not precipitate. The resulting precipitate is discarded. In the next step, the concentration of ammonium sulfate is selected at which the VLP of AP205 is quantitatively precipitated and the VLP of AP205 is isolated from the precipitate formed in this step by centrifugation (14,000 rpm, 20 min). The resulting precipitate was dissolved in NET buffer.

Chromatography:

Capsid protein from the combined supernatants was placed on a 4B column (2.8 × 70 cm) Sepharose column and eluted with NET buffer at 4 ml / hour / fraction. Fractions 28-40 were collected and precipitated with ammonium sulfate at a saturation of 60%. Fractions were analyzed by SDS-PAGE and Western blotting with antiserum specific for AP205 prior to precipitation. The precipitate isolated by centrifugation was redissolved in NET buffer and placed on a 2B Sepharose column (2.3 × 65 cm), eluting at 3 ml / hour / fraction. Fractions were analyzed by SDS-PAGE, and fractions 44-50 were collected, pooled and precipitated with ammonium sulfate at 60% saturation. The precipitate isolated by centrifugation was redissolved in NET buffer and purified on a 6B Sepharose column (2.5 × 47 cm), eluting at 3 ml / hour / fraction. Fractions were analyzed by SDS-PAGE. Fractions 23-27 were collected, the salt concentration was adjusted to 0.5 M and precipitated with PEG 6000 added from 40% mother liquor in water to a final concentration of 13.3%. The precipitate isolated by centrifugation was redissolved in NET buffer and placed on the same column with 2B Sepharose as above, eluting in the same manner. Fractions 43-53 were collected and precipitated with ammonium sulfate at 60% saturation. The precipitate isolated by centrifugation was redissolved in water, and the resulting protein solution was intensively dialyzed against water. It was possible to obtain approximately 10 mg of purified protein per gram of cells.

Examination of virus-like particles by electron microscopy showed that they are identical to phage particles.

Now, after a full description of the present invention in some detail by way of illustration and example for the purpose of clarity of understanding, it will be obvious to a person skilled in the art that the same can be accomplished by modifying or changing the invention in a wide and equivalent range of conditions, compositions and other parameters without affecting the scope of the invention or any particular implementation thereof and that such modifications or changes are intended to be included in the scope of the attached claims.

All publications, patents, and patent applications referred to in this specification indicate the level achieved by those skilled in the art to which this invention belongs and are incorporated by reference to the same extent as if each individual publication, patent or patent the application would be specifically and separately indicated as being incorporated by reference.

Claims (30)

1. A composition intended for use in the treatment of obesity, containing: (a) a core particle with at least one first attachment site, wherein said core particle is a virus-like particle, where preferably said virus-like particle is a recombinant virus-like particle, and (b) at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is ghrelin or ghrelin peptide and wherein said second attachment site is selected from the group consisting of: (i) an attachment site not found in nature with the indicated antigen or antigenic determinant; and (ii) an attachment site found in nature with the indicated antigen or antigenic determinant, wherein said second attachment site is capable of association with said first attachment site and where said ghrelin or ghrelin peptide and said core particle interact through said association to form an ordered and repeating array of antigens. 2. The composition according to claim 1, where the specified virus-like particle is a core antigen of hepatitis B virus or contains recombinant proteins or fragments thereof selected from the group consisting of: (a) recombinant proteins of hepatitis B virus; (b) recombinant measles virus proteins; (c) recombinant Sindbis virus proteins; (d) recombinant rotavirus proteins; (e) recombinant FMD virus proteins; (f) recombinant retrovirus proteins; (g) Norfolk virus recombinant proteins; (h) recombinant alphavirus proteins; (i) recombinant human papillomavirus proteins; (j) recombinant polyoma virus proteins; (k) recombinant bacteriophage proteins; (l) recombinant proteins of RNA phages; (m) recombinant Tu proteins; (n) recombinant proteins of phage Qβ; (o) recombinant proteins of phage GA; (p) recombinant proteins of phage fr; (q) recombinant proteins of phage AP205 and (r) fragments of any of the recombinant proteins from (a) to (q). 3. The composition according to claim 1, where the specified virus-like particle contains or alternatively consists of recombinant RNA phage proteins or fragments thereof, where the specified RNA phage is preferably selected from the group consisting of: (a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (k) bacteriophage f2; (l) bacteriophage PP7; and (m) AP205 bacteriophage. 4. The composition according to claim 1, where the specified virus-like particle contains or alternatively consists of recombinant proteins of RNA phage Qβ, RNA phage fr or RNA phage AP205 or fragments thereof. 5. The composition according to claim 3, where the recombinant proteins contain or alternatively essentially consist or alternatively consist of protein coat RNA phages. 6. The composition of claim 5, wherein said RNA phage coat protein has the amino acid sequence of SEQ ID NO: 4. 7. The composition according to claim 3, where the recombinant proteins contain or alternatively essentially consist or alternatively consist of mutant RNA phage coat proteins, and wherein said RNA phage is preferably selected from the group consisting of: (a) bacteriophage Qβ; (b) bacteriophage R17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP; (f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i) bacteriophage NL95; (k) bacteriophage f2; (l) bacteriophage PP7; and (m) AP205 bacteriophage. 8. The composition of claim 7, wherein said mutant coat proteins of said RNA phage are modified by (i) removing at least one lysine residue by substitution; or (ii) adding at least one lysine residue by substitution; or (iii) deletion of at least one lysine residue; or (iv) adding one lysine residue by insertion. 9. The composition of claim 1, wherein said virus-like particle is a virus-like particle of an RNA phage. 10. The composition of claim 9, wherein said RNA phage is Qβ. 11. The composition of claim 9, wherein said second attachment site is capable of association with said first attachment site via at least one non-peptide covalent bond. 12. The composition according to claim 1, where the specified antigen or antigenic determinant is a ghrelin or ghrelin peptide selected from the group consisting of: (a) human ghrelin or human ghrelin peptide; (b) bovine ghrelin or bovine ghrelin peptide; (c) sheep ghrelin or sheep ghrelin peptide; (d) canine ghrelin or canine ghrelin peptide; (e) feline ghrelin or feline ghrelin peptide; (f) mouse ghrelin or mouse ghrelin peptide; (g) porcine ghrelin or porcine ghrelin peptide; (h) equine ghrelin or equine ghrelin peptide; and (k) a peptide or fragment thereof of any ghrelin from (a) to (h). 13. The composition of claim 12, wherein said antigen or antigenic determinant is a ghrelin peptide and where said ghrelin peptide is a human ghrelin peptide, a dog ghrelin peptide, or a feline ghrelin peptide. 14. The composition of claim 1, wherein said ghrelin peptide contains at least five consecutive amino acids of said ghrelin. 15. The composition according to claim 1, where the specified ghrelin or the specified peptide ghrelin contains an amino acid sequence selected from the group consisting of: (a) GSSFLSPEHQRVQRKESKKPPAKLQPR (SEQ ID NO: 48) (b) GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO: 31) (c) GSSFLSPEHQKLQQRKESKKPPAKLQPR (SEQ ID NO: 49) (d) GSSFLSPEHQKLQRKESKKPPAKLQPR (SEQ ID NO: 50) (e) GSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID NO: 32) (f) GSSFLSPEHQKAQRKESKKPPAKLQPR (SEQ ID NO: 51) (q) KKPPAKLQPR (SEQ ID NO: 52) (h) PPAKLQPR (SEQ ID NO: 53) (i) AKLQPR (SEQ ID NO: 54) (j) GSSFLSPEHQ (SEQ ID NO: 55) (k) EHQRVQQRKE (SEQ ID NO: 56) (l) KLQPR (SEQ ID NO: 59) (m) GSSFLSPEHQRVQ (SEQ ID NO: 60) (n) QRKESKKPPAKLQPR (SEQ ID NO: 61) (o) GSSFLSPEHQKLQ (SEQ ID NO: 62) (p) QRKESKKPPAKLQPR (SEQ ID NO: 63) (q) EHQRVQQRKES (SEQ ID NO: 111) (r) EHQKAQQRKE (SEQ ID NO: 112) (s) EHQKAQQRKES (SEQ ID NO: 113) (t) EHQKLQQRKE (SEQ ID NO: 114) (u) EHQKLQQRKES (SEQ ID NO: 115) (v) LSPEHQRVQQ (SEQ ID NO: 116) (w) LSPEHQKAQQ (SEQ ID NO: 117) (x) LSPEHQKLQQ (SEQ ID NO: 118) and (s) GSSFLSP (SEQ ID NO: 119). 16. The composition according to claim 1, where the specified peptide ghrelin contains the amino acid sequence of SEQ ID NO: 119. 17. The composition according to claim 1, where the specified ghrelin or the specified peptide ghrelin consists of an amino acid sequence selected from the group consisting of: (a) GSSFLSPEHQRVQRKESKKPPAKLQPR (SEQ ID NO: 48) (b) GSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO: 31) (c) GSSFLSPEHQKLQQRKESKKPPAKLQPR (SEQ ID NO: 49) (d) GSSFLSPEHQKLQRKESKKPPAKLQPR (SEQ ID NO: 50) (e) GSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID NO: 32) (f) GSSFLSPEHQKAQRKESKKPPAKLQPR (SEQ ID NO: 51) (g) KKPPAKLQPR (SEQ ID NO: 52) (h) PPAKLQPR (SEQ ID NO: 53) (i) AKLQPR (SEQ ID NO: 54) (j) GSSFLSPEHQ (SEQ ID NO: 55) (k) EHQRVQQRKE (SEQ ID NO: 56) (l) KLQPR (SEQ ID NO: 59) (m) GSSFLSPEHQRVQ (SEQ ID NO: 60) (n) QRKESKKPPAKLQPR (SEQ ID NO: 61) (o) GSSFLSPEHQKLQ (SEQ ID NO: 62) (p) QRKESKKPPAKLQPR (SEQ ID NO: 63) (q) EHQRVQQRKES (SEQ ID NO: 111) (r) EHQKAQQRKE (SEQ ID NO: 112) (s) EHQKAQQRKES (SEQ ID NO: 113) (t) EHQKLQQRKE (SEQ ID NO: 114) (u) EHQKLQQRKES (SEQ ID NO: 115) (v) LSPEHQRVQQ (SEQ ID NO: 116) (w) LSPEHQKAQQ (SEQ ID NO: 117) (x) LSPEHQKLQQ (SEQ ID NO: 118) and (s) GSSFLSP (SEQ ID NO: 119). 18. The composition of claim 1, wherein said second attachment site is capable of association with said first attachment site via at least one non-peptide covalent bond. 19. The composition of claim 1, further comprising an amino acid linker, wherein said amino acid linker comprises or alternatively consists of said second attachment site, and where said second attachment site or said amino acid linker with said second attachment site is linked to said ghrelin or said ghrelin peptide at its C-terminus or alternatively at its N-terminus and said second attachment site or said amino acid linker with said second attachment site is selected and h group consisting of: (a) GGC; (b) GGC-CONH ₂ , (c) GC; (d) GC-CONH ₂ , (e) C; and (f) C-CONH ₂ . 20. The composition of claim 1, wherein said ghrelin or said ghrelin peptide with said at least second attachment site consists of an amino acid sequence selected from the group consisting of: (a) CGSSFLSPEHQRVQRKESKKPPAKLQPR (SEQ ID NO: 64) (b) CGSSFLSPEHQRVQQRKESKKPPAKLQPR (SEQ ID NO: 65) (c) CGSSFLSPEHQKLQQRKESKKPPAKLQPR (SEQ ID NO: 71) (d) CGSSFLSPEHQKLQRKESKKPPAKLQPR (SEQ ID NO: 72) (e) CGSSFLSPEHQKAQQRKESKKPPAKLQPR (SEQ ID NO: 77) (f) CGSSFLSPEHQKAQRKESKKPPAKLQPR (SEQ ID NO: 106) (g) GSSFLSPEHQRVQRKESKKPPAKLQPRC (SEQ ID NO: 66) (h) GSSFLSPEHQRVQRKESKKPPAKLQPRGC (SEQ ID NO: 120) (i) GSSFLSPEHQRVQQRKESKKPPAKLQPRC (SEQ ID N0: 67) (j) GSSFLSPEHQRVQQRKESKKPPAKLQPRGC (SEQ ID NO: 121) (k) GSSFLSPEHQKLQQRKESKKPPAKLQPRC (SEQ ID NO: 73) (l) GSSFLSPEHQKLQQRKESKKPPAKLQPRGC (SEQ ID NO: 123) (m) GSSFLSPEHQKLQRKESKKPPAKLQPRC (SEQ ID NO: 74) (n) GSSFLSPEHQKLQRKESKKPPAKLQPRGC (SEQ ID NO: 124) (o) GSSFLSPEHQKAQQRKESKKPPAKLQPRC (SEQ ID NO: 105) (p) GSSFLSPEHQKAQRKESKKPPAKLQPRC (SEQ ID NO: 107) (q) CKKPPAKLQPR (SEQ ID NO: 108) (r) CPPAKLQPR (SEQ ID NO: 70) (s) CAKLQPR (SEQ ID NO: 109) (t) GSSFLSPEHQC (SEQ ID NO: 110) (u) CEHQRVQQRKE (SEQ ID NO: 76) (v) GSSFLSPEHQRVQC (SEQ ID NO: 68) (w) GSSFLSPEHQRVQGC (SEQ ID NO: 122) (x) CQRKESKKPPAKLQPR (SEQ ID NO: 69) (s) GSSFLSPEHQKLQC (SEQ ID NO: 75) (z) GSSFLSPEHQKLQGC (SEQ ID NO: 125) (aa) GSSFLSPEHQKAQRKESKKPPAKLQPRC (SEQ ID NO: 126) (bb) GSSFLSPEHQKAQRKESKKPPAKLQPRGC (SEQ ID NO: 127) (cc) GSSFLSPEHQKAQQRKESKKPPAKLQPRC (SEQ ID NO: 128) (dd) GSSFLSPEHQKAQQRKESKKPPAKLQPRGC (SEQ ID NO: 129) (her) GSSFLSPEHQKAQC (SEQ ID NO: 130) (ff) GSSFLSPEHQKAQGC (SEQ ID NO: 131) (gg) GGSSFLSPEHQGC (SEQ ID NO: 132) (hh) CKKPPAKLQPR (SEQ ID NO: 133) (ii) CEHQKAQQRKE (SEQ ID NO: 134) (jj) CEHQKAQQRKES (SEQ ID NO: 135) (kk) CLSPEHQKAQQ (SEQ ID NO: 136) (ll) CEHQRVQQRKES (SEQ ID NO: 137) and (mm) CLSPEHQRVQQ (SEQ ID NO: 138). 21. The composition according to claim 1, where the first attachment site is a lysine residue, and the second attachment site is a cysteine residue. 22. The composition according to claim 1, where the specified antigen or antigenic determinant is a biologically inactive form of ghrelin or preferably ghrelin peptide that does not contain modifications of n-octanoyl. 23. The composition according to claim 1, where the specified ghrelin or ghrelin peptide contains n-octanoylation at the serine residue, where the specified serine residue corresponds to the serine residue at position 3 of ghrelin. 24. A pharmaceutical composition for use in the treatment of obesity, comprising: (a) the composition according to claim 1 and (b) an acceptable pharmaceutical carrier, and where preferably said pharmaceutical composition further comprises an adjuvant, or where preferably said pharmaceutical composition does not contain an adjuvant. 25. A vaccine composition intended for use in the treatment of obesity, containing the composition according to claim 1. 26. The vaccine composition according A.25, where the specified vaccine composition does not contain adjuvant. 27. The vaccine composition of claim 25, wherein said vaccine composition further comprises an adjuvant. 28. An immunization method comprising administering to an animal or human a composition according to any one of the preceding paragraphs, wherein preferably said antigen or antigenic determinant is an autoantigen. 29. The immunization method of claim 28, wherein (i) said animal is a human and where said antigen or antigenic determinant is human ghrelin or ghrelin peptide; or (ii) where said animal is a feline and where said antigen or antigenic determinant is ghrelin or ghrelin peptide; or (iii) where said animal is an animal of the canine family and where said antigen or antigenic determinant is a feline ghrelin or a ghrelin peptide. 30. A method of treating obesity, comprising administering a composition according to any one of claims 1 to 23, a vaccine composition according to any one of claims 25 to 27, or a pharmaceutical composition according to claim 24 to an animal, preferably a human, cat or dog.

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